Small-diameter acellular porcine corneal stroma for peripheral corneal ulceration treatment

AIM:To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma(SAPS)for the treatment of peripheral corneal ulceration(PCU).METHODS:This retrospective clinical study included 18 patients(18 eyes)with PCU between April 2018 and December 2020.All patients had PCU and underwent lamellar keratoplasty with SAPS.Observation indicators included preoperative and postoperative best-corrected visual acuity(BCVA)and transparency of SAPS.The infection control rate in the surgical eye-lesion area was also calculated.RESULTS:Eighteen patients underwent lamellar keratoplasty with SAPS to treat PCU.None of the patients experienced rejection after 6mo(18/18)and 12mo(16/16)of follow-up.The BCVA(0.47±0.30)at the 6mo followup after operation was significantly improved compared with the baseline(0.99±0.80),and the difference was statistically significant(Z=-3.415,P<0.05).The BCVA at the 12mo follow-up after operation was not statistically significant compared to the 6mo(Z=0,P=1).With time,the SAPS graft gradually became transparent.At the 6mo(18/18)and 12mo(16/16)follow-up,none of the patients had recurrent corneal infection.CONCLUSION:SAPS is clinically effective in the treatment of PCU,improving the patient’s BCVA and reducing the incidence of rejection after keratoplasty.

Intraocular pressure before and after capsulorhexis using two viscoelastic substances and two surgical approaches in enucleated porcine eyes

AIM:To investigate the influence of ophthalmic viscoelastic devices(OVDs)and different surgical approaches on the intraocular pressure(IOP)before and after creation of the curvilinear circular capsulorhexis(CCC)as a measure for anterior chamber stability during this maneuver.METHODS:Prospective experimental WetLab study carried out on enucleated porcine eyes.IOP was measured before and after CCC with the iCare Rebound tonometer(iCare ic200;iCare Finland Oy,Vantaa,Finland).The OVDs used were a cohesive one[Z-Hyalin,Carl Zeiss Meditec AG,Germany;hyaluronic acid(HA)]and a dispersive[Z-Celcoat,Carl Zeiss Meditec AG,Germany;hydroxy propylmethylcellulosis(HPMC)].The CCC was created using Utrata forceps or 23 g microforceps in different combinations with the OVDs.RESULTS:Using the Utrata forceps the IOP dropped from 63.65±6.44 to 11.25±3.63 mm Hg during the CCC.The use of different OVDs made no difference.Using the 23 g microforceps the IOP dropped from 65.35±8.15 to 36.55±6.09 mm Hg.The difference between IOP drop using either Utrata forceps or 23 g microforceps was highly significant regardless of the OVD used.CONCLUSION:Using the sideport for the creation of the capsulorhexis leads to a lesser drop in IOP during this maneuver compared to the main incision in enucleated porcine eyes.The use of different OVD has no significant influence on IOP drop.

Porcine enteric alphacoronavirus infection increases lipid droplet accumulation to facilitate the virus replication

Coronaviruses are widely transmissible between humans and animals, causing diseases of varying severity. Porcine enteric alphacoronavirus(PEAV) is a newly-discovered pathogenic porcine enteric coronavirus in recent years, which causes watery diarrhea in newborn piglets. The host inflammatory responses to PEAV and its metabolic regulation mechanisms remain unclear, and no antiviral studies have been reported. Therefore, we investigated the pathogenic mechanism and antiviral drugs of PEAV. The transcriptomic analysis of PEAV-infected host cells revealed that PEAV could upregulate lipid metabolism pathways. In lipid metabolism, steady-state energy processes, which can be mediated by lipid droplets(LDs), are the main functions of organelles. LDs are also important in viral infection and inflammation. In infected cells, PEAV increased LD accumulation, upregulated NF-κB signaling, promoted the production of the inflammatory cytokines IL-1β and IL-8, and induced cell death. Inhibiting LD accumulation with a DGAT-1 inhibitor significantly inhibited PEAV replication, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death. The NF-κB signaling pathway inhibitor BAY11-7082 significantly inhibited LD accumulation and PEAV replication. Metformin hydrochloride also exerted anti-PEAV effects and significantly inhibited LD accumulation, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death. LD accumulation in the lipid metabolism pathway therefore plays an important role in the replication and pathogenesis of PEAV, and metformin hydrochloride inhibits LD accumulation and the inflammatory response to exert anti-PEAV activity and reducing pathological injury. These findings contribute new targets for developing treatments for PEAV infections.

Enhancement of porcine in vitro embryonic development through luteolin‑mediated activation of the Nrf2/Keap1 signaling pathway

Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.

Identification,Isolation and Genomic Characterization of Porcine Astrovirus in Shandong Province,China in 2021-2023

[Objective]The paper was to identify,isolate,and characterize porcine astrovirus in Shandong Province between 2021 and 2023.[Method]A total of 1025 samples of porcine diarrhea samples were collected from various regions of Shandong Province between January 2021 and October 2023.The samples were tested by RT-PCR,followed by sequencing and phylogenetic analyses of the polymerase.[Result]The total positive rate of PAstV was 34.6%(355/1025).The respective proportions of individuals infected with PAstV-1,PAstV-2,PAstV-4 and PAstV-5 were 25.4%(90/355),28.2%(100/355),35.2%(125/355)and 22.5%(80/355),respectively.Additionally,mixed infection was observed.Meanwhile,849 samples of healthy pigs were tested by RT-PCR,and the results demonstrated that the total positive rate of PAstV was 8.13%(69/849).Of these,the proportion of PAstV-1,PAstV-2 and PAstV-4 infection was 27.5%(19/69),37.7%(26/69)and 40.6%(28/69),and a mixed infection also existed.Further sequencing and characterization of some the selected isolates revealed low sequence identities(56.2%)with known PAstV strains,indicating the presence of novel types or genotypes of PAstVs.Furthermore,the isolation conditions of porcine astrovirus were optimized,resulting in the purification of a pure PAstV-4 strain(designated PAstV-4-GRF1).The virus was found to exhibit typical astroviral morphology,with nucleotide identity ranging from 89.9 to 95.4%with previously published PAstV-4 strains.Then,macrovirus transcriptome sequencing showed that 88.30%of the CRF1 samples were mammalian astroviruses.By species classification,PAstV 4 and PAstV 2 accounted for 21.79%and 0.32%,respectively.Phylogenetic tree analysis showed that the c15050 fragment was identical to the GRF-1 sequencing fragment of the isolated strain,and exhibited the highest homology with the Hunan PAstV-4 sequence MK460231 in China.[Conclusion]As the inaugural isolated PAstV-4 strain,it furnishes pivotal materialfor the investigation ofthe biological and pathogenic properties of this virus as well as for the prospectivedevelopment of relevant biological and diagnostic reagents.

Interlayer repair with porcine small intestinal submucosa versus internal repair with tragus cartilage in endoscopic tympanoplasty

Objective Endoscopic tympanoplasty includes various surgical methods,such as internal repair,interlayer repair,and external overlay.This technique requires autologous materials,allografts,and xenografts,which are used to repair tympanic membrane(TM)perforation.To obtain good results,appropriate surgical methods and repair materials should be selected.This study aims to assess the efficacy of repairing refractory TM perforations in the porcine small intestinal submucosa(SIS)during transcanal endoscopic type I tympanoplasty.Method A retrospective chart review was performed on patients who underwent TM perforation repair with porcine SIS and tragus cartilage between January 2022 and September 2022 at Sir Run Run Shaw Hospital,Zhejiang University School of Medicine.Perforation size,tympanic status,pre-and postoperative symptoms,follow-up data,wound healing rates,and hearing improvement were analysed.Results Of the 115 patients included in the study,56 underwent interlayer repair with porcine SIS of the TM,and 59 patients underwent internal repair with tragus cartilage.No significant difference was found between the two groups at baseline in terms of age,sex,disease course,perforation side,tympanic status,underlying disease,or preoperative infection.The total postoperative effective rate of interlayer implantation with porcine SIS was 91.07%(51 patients),and that of internal implantation with tragus cartilage was 88.14%(52 patients).No significant difference was found in terms of the graft success rate between the two surgical methods(p=0.887).Postoperative pure tone auditory(PTA)and air-bone gap(ABG)density significantly increased in both groups compared with before surgery(p<0.05).However,the postoperative PTA and ABG density were not significantly different 3 months post-surgery between the two groups(p>0.05).Compared to those in the internal implantation group,the patients in the interlayer group had a shorter operation duration(51.36±6.76 min vs.59.71±7.45 min,t=6.298,p<0.001)and less blood loss(11.91±2.61 mL vs.15.27±2.57 mL,t=7.019,p<0.001).Conclusions Our study suggests that the porcine SIS,as well as the tragus cartilage,has a high success rate in repairing irreversible TM perforation.Endoscopic tympanoplasty via interlayer implantation with porcine SIS offers distinct advantages,including the absence of donor-site incision and scar formation,and ease of graft modification and manipulation.

Acute Toxicity of Recombinant Porcine Interferon-alpha

To observe the acute toxicity of recombinant porcine interferen-alpha （IFN-alpha） in mice and thus provide a basis for the clinical safety. [Method] According to the principles of acute toxicity, all the mice were divided into two major groups （intraperitoneally injected group and intramuscularly injected group） respectively at high dose, moderate dose and low dose. And the normal control group was also set up. Within 14 d after administration, the behavior of mouse and the degree of toxicity were continuously observed. The hematological indexes and biochemical indexes of blood were detected to obtain the preliminary toxicity data of the recombinant porcine IFN-alpha. And at the end of the experiment, mice were sacrificed for autopsy. [ Result] There was not significant difference in external performance, behavioral characteristics, body temperature, weight, pathological anatomy of visceral organs, hematological indexes and biochemical indexes between the experimental groups and the control group. [ Conclusion] The highest dose of porcine interferon （5.0 x 10s IU per mouse） in this experiment or the dose lower than this dosage should not have significant toxic effects on mice, and the recombinant porcine IFN-alpha is safe in clinical application.

Isorhamnetin protects porcine oocytes from zearalenone-induced reproductive toxicity through the PI3K/Akt signaling pathway

Background Zearalenone(ZEA)widely exists in moldy grains,which seriously destroys the fertility of females.Isorhamnetin,a natural flavonoid,has extensive of pharmacological activities.However,the beneficial effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced porcine oocyte damage have not been investigated.Methods Oocytes were treated with different concentrations of ZEA(3,5,8 and 10μmol/L)and isorhamnetin(5,10,20 and 30μmol/L)for 44 h at 39℃.ZEA(5μmol/L)and isorhamnetin(10μmol/L)were selected for subsequent studies.Polar body exclusion rate,apoptosis rate and apoptosis related proteins,ROS levels and SOD2 protein,mitochondrial membrane potential and distribution,endoplasmic reticulum distribution and proteins expression,and PI3K,Akt and p-Akt proteins expression of oocytes were detected.In addition,the effect of PI3K antagonist(LY294002)on oocyte nuclear maturation and apoptosis were used to determine the involvement of PI3K/Akt signaling pathway.Results Our findings showed that ZEA exposure damaged oocytes and isorhamnetin therapy restored the developmental capability of porcine oocytes.Isorhamnetin promoted polar body extrusion rate to rescue ZEA-induced meiotic arrest in porcine oocytes.Isorhamnetin alleviated ZEA-induced oxidative stress by stimulating SOD2 protein expression and inhibiting ROS production.Moreover,isorhamnetin enhanced normal mitochondrial distribution and mitochondrial membrane potential to prevent mitochondrial dysfunction induced by ZEA.Changing the expression of endoplasmic reticulum stress-related marker proteins(CHOP,GRP78)and the distribution rate of normal endoplasmic reticulum showed that isorhamnetin relieved ZEA-caused endoplasmic reticulum stress.Mechanistically,isorhamnetin decreased Bax/Bcl-2 protein expression and inhibited ZEA-induced apoptosis through PI3K/Akt signaling pathway.Conclusions Collectively,these results suggest that isorhamnetin protects oocytes from ZEA-caused damage through PI3K/Akt signaling pathway,which enhances meiotic maturation and mitochondrial function,and inhibits early apoptosis,oxidative stress and endoplasmic reticulum stress in porcine oocytes.Our study provides a new strategy for solving the reproductive toxicity induced by ZEA and treating woman infertility.

Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome(PRRS)is recognized as one of the most infectious viral diseases of swine.Although Cluster of differentiation 163(CD163)is identified as an essential receptor for mediating PRRS virus(PRRSV)infection,the important residues involved in infection on CD163 are still unclear.Therefore,it is very important to identify these key residues to study the mechanism of PRRSV infection and to generate anti-PRRSV pigs.In this study,we first generated immortalized porcine alveolar macrophage(IPAM)cell lines harboring 40-residues(residues 523-562,including R561(arginine(R)at position 561))deletion of CD163.PRRSV infection experiments showed that these IPAM cell lines were completely resistant to PRRSV infection.We then generated cloned pigs carrying CD163-R561A(an arginine(R)to alanine(A)substitution at position 561 of CD163).PRRSV challenge experiments in porcine alveolar macrophages(PAMs)isolated from the CD163-R561A pigs showed significantly lower susceptibility to PRRSV than that of CD163-R561 PAMs.Through this study,we show that CD163523-562 contains essential residues for mediating PRRSV infection,and that CD163 R561 significantly contributes to PRRSV infection but is not essential for infection.These functional sites can therefore serve as new targets for understanding the mechanism of PRRSV infection.Furthermore,CD163-R561A pigs can be used as an important model for improving pig germplasm with resistance against PRRSV.

A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus,African swine fever virus,and atypical porcine pestivirus

With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results were the same(100%).A total of 451 clinical samples were detected,and the results showed that the positive rates of CSFV,ASFV,and APPV were 0.22% (1/451),1.3%(6/451),and 0%(0/451),respectively.This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV,ASFV,and APPV.

Dynamic compressive response of porcine muscle measured using a split Hopkinson bar system with a pair of PVDF force transducers

The classic metallic Split Hopkinson Pressure Bar(SHPB)cannot capture the transmitted signal accurately when measuring soft biological tissue,because of the very low wave impedance and strength of this material.So the dynamic compressive response of porcine muscle has been investigated by using a modified SHPB.The forces on both ends of the sample measured using Polyvinylidene fluor(PVDF)transducers were applied to calculate the stress in the specimen instead of the strain gauge signal on the transmitted bar.Moreover,a circular cardboard disk pulse shaper was applied for generating a suitable incident pulse to achieve stress equilibrium and constant strain rates in the specimens.Then,the dynamic mechanical properties of porcine muscle parallel and perpendicular to the fiber directions were measured,and the stress equilibrium process during loading was analyzed,as well as the inertia-induced extra stress being corrected.Furthermore,quasi-static tests were conducted at two different strain rates to investigate the strain rate dependence using a universal material testing machine.The results show that the stress-strain curves are sensitive to strain rate in the two different loading directions.The compressive stress perpendicular to the fiber direction is stiffer than that parallel to the fiber direction.In addition,a strain rate-dependent constitutive model was developed based on the mechanical response of the muscle at different strain rates and fitted to the experimental data.The results show that the overall fit is good,and the constitutive model could describe the muscle's dynamic mechanical properties.

Anethole improves the developmental competence of porcine embryos by reducing oxidative stress via the sonic hedgehog signaling pathway

Background Anethole(AN)is an organic antioxidant compound with a benzene ring and is expected to have a positive impact on early embryogenesis in mammals.However,no study has examined the effect of AN on porcine embryonic development.Therefore,we investigated the effect of AN on the development of porcine embryos and the underlying mechanism.Results We cultured porcine in vitro-fertilized embryos in medium with AN(0,0.3,0.5,and 1 mg/mL)for 6 d.AN at 0.5 mg/mL significantly increased the blastocyst formation rate,trophectoderm cell number,and cellular survival rate compared to the control.AN-supplemented embryos exhibited significantly lower reactive oxygen species levels and higher glutathione levels than the control.Moreover,AN significantly improved the quantity of mitochondria and mitochondrial membrane potential,and increased the lipid droplet,fatty acid,and ATP levels.Interestingly,the levels of proteins and genes related to the sonic hedgehog(SHH)signaling pathway were significantly increased by AN.Conclusions These results revealed that AN improved the developmental competence of porcine preimplantation embryos by activating SHH signaling against oxidative stress and could be used for large-scale production of high-quality porcine embryos.

Associations between maternal vitamin D status and porcine litter characteristics throughout gestation

Emerging evidence suggests an important role of vitamin D in the establishment and maintenance of pregnancy,and the regulation of foetal growth across mammalian species.However,the temporal changes in maternal vitamin D sta-tus throughout gestation in the pig and the relationship between maternal vitamin D status and litter characteristics of interest across gestation remain poorly understood and under-investigated.The abundance of 25(OH)D in maternal plasma was quantified by HPLC–MS/MS at gestational days(GD)18,30,45,60 and 90(n=5–11 gilts/GD).Maternal plasma 25(OH)D concentrations significantly increased between GD18 and GD30(P<0.05).The relationship between maternal vitamin D metabolite concentrations and litter characteristics of interest including gilt weight,ovulation rate,mean litter weight,number of live foetuses,percentage prenatal survival,and sex ratio of the litter was assessed.Maternal 25(OH)D(P=0.059)concentrations tended to be positively associated with percentage prenatal survival on GD60.On GD90,maternal 25(OH)D(P<0.05)concentrations were inversely associated with gilt weight.Maternal plasma 25(OH)D concentrations were inversely associated with the percentage of male foetuses in the litter on GD90(P<0.05).This study has provided novel insights into temporal changes in maternal vitamin D status throughout ges-tation and the relationship between maternal vitamin D status and the economically important litter characteristics of gilt weight,percentage prenatal survival and percentage of male foetuses in the litter.Improving the understanding of the role of vitamin D across important developmental timepoints in relation to foetal growth is essential to improve reproductive success in livestock species.

Comparison of emergency surgical cricothyroidotomy and percutaneous cricothyroidotomy by experienced airway providers in an obese,in vivo porcine hemorrhage airway model

Background: Emergency front-of-neck airway(eFONA) is a life-saving procedure in “cannot intubate, cannot oxygenate”(CICO). The fastest and most reliable method of eFONA has not been determined. We compared two of the most advocated approaches: surgical cricothyroidotomy and percutaneous cricothyroidotomy, in an obese, in vivo porcine hemorrhage model, designed to introduce real-time physiological feedback, relevant and high provider stress. The primary aim was to determine the fastest method to secure airway. Secondary aims were arterial saturation and partial pressure of oxygen, proxy survival and influence of experience.Methods: Twelve pigs [(60.3±4.1) kg] were anesthetized and exposed to 25%–35% total blood volume hemorrhage before extubation and randomization to Seldinger technique “percutaneous cricothyroidotomy”(n=6) or scalpelbougie-tube technique “surgical cricothyroidotomy”(n=6). Specialists in anesthesia and intensive care in a tertiary referral hospital performed the eFONA, simulating an actual CICO-situation.Results: In surgical cricothyroidotomy vs. percutaneous cricothyroidotomy, the median(interquartile range, IQR) times to secure airway were 109(IQR 71–130) s and 298(IQR 128–360) s(P=0.0152), arterial blood saturation(SaO2) were 74.7(IQR 46.6–84.2)% and 7.9(IQR 4.1–15.6)%(P=0.0167), PaO_(2) were 7.0(IQR 4.7–7.7) kPa and 2.0(IQR 1.1–2.9) kPa(P=0.0667), and times of cardiac arrest(proxy survival) were 137–233 s, 190(IQR 143–229) s, from CICO. All six animals survived surgical cricothyroidotomy, and two of six(33%) animals survived percutaneous cricothyroidotomy. Years in anesthesia, 13.5(IQR 7.5–21.3), did not influence time to secure airway.Conclusions: eFONA by surgical cricothyroidotomy was faster and had increased oxygenation and survival, when performed under stress by board certified anesthesiologists, and may be an indication of preferred method in situations with hemorrhage and CICO, in obese patients.

Purification of Recombinant Porcine Interferon-Alpha

[ Objective] To study the purification of recombinant porcine interferon-alpha （rPolFN-alpha） and lay a foundation for researches on the structure of the rPolFN-alpha and the preparation of standard proteins. [ Method] The rPolFN-alpha were induced and extracted from the recombi- nant E. coil BL21, and they were purified by two strategies. The first strategy was that the rPolFN-alpha were purified by GST （ glutathione S transferase） affinity chromatography, DEAE （diethylaminoethyl） anion exchange chromatography and gel filtration in turn defined as three-step chromatography method; the second strategy was that the rPolFN-alpha were purified by GST affinity chromatography and gel filtration in tum defined as two-step chromatography method. Then the purified products were detected by the SDS-PAGE （sodium dodecyl sulfate polyacrylamide gel electrophoresis） and were identified by western-blotting. [Result] The purity quotient of pudfied products of the two-step chromatography method was 96.0% and that of the three-step chromatography method was 98.8%. The purified products were detected by the SDS-PAGE and the western- blotting, respectively. The results showed that the target band was 45.0 kDa and the specific band was found. [ Conclusion] The purity quotient of proteins of the two-step chromatography method is close to that of the three-step chromatography method, thus the two-step chromatography meth- od is more convenient and more suitable for pilot production than the three-step chromatography method.

Development of Multi-PCR for Differentiation of Vaccine Strain and Field Isolate of Porcine Pseudorabies Virus

Three pairs of primer were designed for amplification of porcine pseudorabies virus （PRV） gB, gE, and TK gene by multiplex PCR （multi-PCR） in order to differentiate vaccine strains from field isolates. Three specific bands were obtained respectively at the expected size, 427 bp （gB gene）, 298 bp （gEgene）, and 208 bp （TKgene）, Then four different gene-deleted vaccines of PRV were detected by multi-PCR. One ex- pected specific band was observed in one of samples, while two bands in the others. As shown by the detection results, the multi-PCR has high sensitivity and specificity and should be applied in pathogen diagnosis and epidemiological investigation in the future.

Expression of Porcine Reproductive and Respiratory Syndrome Virus ORF7 Gene and Purification and Immunological Activity Analysis of the Recombinant Protein

[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 （DE3） and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 （DE3）. Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.

Location of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus in Tissues of Natural Cases

[ Objective] The aim of this study was to provide a theoretical basis for the prevention and treatment of highly pathogenic porcine reproductive and respiratory syndrome （HP-PRRS）. [Method] Antigen location and histopathological observation in natural cases infected by highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） were analyzed by immunohistochemistry and H. E. staining. [Result] The virus antigen mainly existed in epithelial calls, and also a few in mecrophages, lymphocytes and brain nerve cells. [ Conclusion] The cell and tissue tropism of HP-PRRSV strain in natural cases is different from that of previous strains.

Purification and Immunocompetence Analysis of GP5 Protein in Porcine Reproductive and Respiratory Syndrome

[Objective] The purification and immunocompetence of GPS protein in porcine reproductive and respiratory syndrome (PRRS) were analyzed in this study, which provided basis for establishing the corresponding serological method. [Method] The recombinant expression plasmid pGEX-6P-5 was transformed into BL21 and expressed after being induced with IPTG. The solubility analysis of expression products was carried out, and then the recombinant protein was purified for SDS-PAGE identification and Western-blot analysis. Finally, the recombinant antigen was used in the immune experiment of guinea pigs. [Result] The target protein content accounted for 30% of the total cells protein content according to the chromatography scanning, and the purity of target protein after purification reached 80%. The purified protein was analyzed by Western-blot and immune experiment of guinea pigs, and the results showed that the expressed protein had good reactionogenicity and immunogenicity. [Conclusion] This study provides materials for further studies on the function between PRRSV ORF5 gene and its editing protein, which also lays a foundation for porcine reproductive and respiratory syndrome virus genetic engineering products.

Study on Cloning and Polymorphism of Porcine Adiponectin Promoter

[ Objective] The aim of this study was to provide a basis for study on adiponectin as a candidate gene for fat deposition. [ Method] The promoter sequence of adiponectin was obtained by porcine BAC library screening and primer-walking method. The polymorphisms of adiponectin promoter from 290 pigs, including 5 breeds of Lantang pig, Large spotted pig, Large white pig, Landrace and Duroc, were analyzed with PCR-RFLP. [ Result] At SNP site of adiponectin 5＇-flanking region -1 010 bp （G/A）, GG genotype frequency in Chinese indigenous pigs was significantly higher than that in exotic pigs. At SNP site of adiponectin 5＇-flanking region -394 bp （T/C）, the genotype distribution of Chi- nese indigenous pigs was abundant, while no CC genotype was detected in exotic pigs, and T allele frequency was higher in exotic pigs. [ Conclusion] SNP site mutation of - 1 010 bp （G/A） may lead to changes of the gene transcription level, while SNP site of -394 bp （T/C） properly has no relationship with gene transcription level and fat deposition.