新时代新农科国际视野本科人才培养模式创新——以四川农业大学动物医学类专业的实践为例

针对“培养质量与社会需求、与新农科建设发展趋势、与国际视野衔接不够甚至部分脱节”等重大课题,围绕构建“情系人民健康的动物医学特色思政教育体系”“新时代新农科动物医学教育全球化师资体系”“面向社会多元需求关键要素协同育人体系”“面向全球动物医学教育一体化课程结构体系”“面向生产实践创新能力的实训教学体系”和“面向社会经济发展的教学质量保障体系”等六大体系支撑的人才培养模式,经20余年不间断的改革和实践,并取得了显著成效。

黄病毒E蛋白N-糖基化研究进展

糖基化是蛋白质一种重要的翻译后修饰调控机制。在真核细胞中,糖基化的合成主要包括:N糖基化、O糖基化及糖基磷脂酰肌醇锚3种途径。新生肽链合成时或合成后,糖链与肽链中特定序列(Asn-X-Thr/Ser,其中X为除脯氨酸外的任意氨基酸)的天冬酰胺(N)相连接,所以将该糖基化类型称为N连接糖链,又称N糖基化[1]。在真核细胞中,N糖基化的合成在内质网和高尔基体中完成,糖蛋白中N连接聚糖在内质网中将预先生成的寡糖转运至新生肽链的天冬酰胺残基中,再将其转移至高尔基体内进一步修饰,并在高尔基体糖苷酶和糖基转移酶的作用下.

Role of duck plague virus glycoprotein C in viral adsorption:Absence of specific interactions with cell surface heparan sulfate

Many mammalian herpes viruses utilize heparan sulfate （HS） moieties present on cell surface proteoglycans as receptors for cell entry, and this process also requires viral glycoprotein C （gC） homologues. However, our understanding of the role of gC in facilitating attachment of other alpha-herpes viruses such as the duck plague virus （DPV） remains preliminary. To study the role of gC during DPV infection, we used a gC-deleted mutant virus （DPV-AgC-EGFP）. Examination of the viral copy number by real-time PCR, as well as time course studies of viral adsorption and proliferation revealed that gC was involved in the viral binding to the cell surface. The affinity of viral glycoproteins （gB-DPV, gC-DPV, and gE-DPV） to HS was assessed using a prokaryotic expression system and HJTrapTM HeparJn HP column chromatography. In addition, to confirm that gC played a role in the interaction between DPV and HS, viruses were treated with the HS analogue heparin and host cells were treated with its inhibitors heparinase prior to exposure to DPV-△gC-EGFP or wild-type strain Chinese virulent duck plague virus （DPV-CHv）. The effects of heparin and heparinase on virus infectivity demonstrated that function of gC on Viral adsorption is independent of interactions between gC and heparin sulfate on cell surface. All in all, this study demonstrated that the gC of DPV can mediate viral adsorption in an HS-independent manner, which distinguish it from the gC of some other alpha-herpes viruses. Future studies will be required to identify the receptors involved in gC protein binding to cells. This work provides us a foundation for further studies of examining the roles of gC in the adsorption during duck plague virus infection.

Acute and subchronic toxicity as well as evaluation of safety pharmacology of modified pulsatilla granules

The present study investigated acute and subchronic toxicity and safety pharmacology of modified pulsatilla granules（MPG）to provide a basis for a comprehensive understanding of MPG toxicity.The results of acute toxicity testing showed that the median lethal dose of MPG was more than 5 000 mg kg^-1,suggesting that MPG was considered as practically non-toxic.The subchronic toxicity study for 30 days was conducted by daily oral administration at doses of 375,750 and 1 500 mg kg^-1 in Sprague-Dawley rats.The results of subchronic toxicity study showed that the body weight and relative organ weight were not significantly changed by administration of MPG.The clinical chemistry study showed that MPG could induce kidney and liver damages.In histopathological,mild lesions in liver and kidney were also observed,suggesting that the liver and kidney might be potential target organs of MPG.In the safety pharmacology study,MPG did not exhibited any side effects to rats in cardiovascular system,respiratory system and central nervous system.These results suggested that MPG could be considered safe for veterinary use.

Antibacterial Activity of 9-Octadecanoic Acid-Hexadecanoic Acid-Tetrahydrofuran-3,4-Diyl Ester from Neem Oil

The 9-octadecanoic acid-hexadecanoic acid-tetrahydrofuran-3,4-diyl ester from neem oil was investigated for antibacterial activity against three bacterial strains viz., Staphylococcus aureus ATCC No. 25923, Escherichia coli ATCC No. 44102 and Salmonella sp. ATCC No. 50 041 in vitro. The minimum inhibitory concentration （MIC） and minimum bactericidal concentration （MBC） values of the 9-octadecanoic acid-hexadecanoic acid-tetrahydrofuran-3,4-diyl ester were determined by using the broth microdilution dilution （BMD） method at different concentrations ranging from 20 to 0.625 mg mL-1. Its time-inhibition curve against E. coli was also tested and showed that the MIC values for the bacterial strains S. aureus, E. coli and Salmonella sp. were 20, 5 and 10 mg mL-1, respectively. Its MBC values were 20, 20 and 10 mg mL-1, respectively. The antibacterial activity of 9-octadecanoic acid-hexadecanoic acid-tetrahydrofuran-3,4-diyl ester against three strain tested showed the relationship with time and concentration.

Alphaherpesvirus-vectored vaccines against animal diseases: Current progress

Recombinant virus-vectored vaccines are novel agents that can effectively activate specific and nonspecific immunity,are multivalent and multieffective,and have high safety ratings.Animal alphaherpesviruses have a large genome,contain multiple nonessential regions that do not affect viral replication and are capable of accepting the insertion of an exogenous gene and expressing the antigen protein.Furthermore,animal alphaherpesviruses have a wide host spectrum,can replicate in the host and continuously stimulate the animal to produce immunity to the corresponding pathogen,thus making them ideal carriers for recombinant virus-vectored vaccines.With the development of gene-editing technology,recombinant viruses capable of expressing foreign genes can be constructed by various methods.Currently,studies on recombinant virusvectored vaccines constructed based on animal alphaherpesviruses have involved poultry,pigs,cattle,sheep,and companion animals.Studies have shown that the construction of recombinant animal alphaherpesviruses enables the acquisition of immunity to multiple diseases.This article mainly summarizes the current progress on animal alphaherpesvirus-vectored vaccines,aiming to provide reference for the development of new animal alphaherpesvirus-vectored vaccines.

Immunogenicity and protective efficacy of DHBV DNA vaccines expressing envelope and capsid fusion proteins in ducks delivered by attenuated Salmonella typhimurium

Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

The codon-optimized capsid gene of duck circovirus can be highly expressed in yeast and self-assemble into virus-like particles

The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid （Opt-Cap） gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% （v/v） methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What＇s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles （VLPs）. These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.

Kaempferol inhibits Pseudorabies virus replication in vitro through regulation of MAPKs and NF-κB signaling pathways

Pseudorabies virus(PRV),in the family Herpesviridae,is a pathogen of Aujeszky’s disease,which causes great economic losses to the pig industry.Recent outbreaks of Pseudorabies imply that new control measures are urgently needed.The present study shows that kaempferol is a candidate drug for controlling PRV infection,as it possesses the ability to inhibit PRV replication in a dose-dependent manner in vitro.Kaempferol at a concentration of 52.40μmol L^(-1) could decrease PRV-induced cell death by 90%.With an 50%inhibitory concentration(IC50)value of 25.57μmol L^(-1),kaempferol was more effective than acyclovir(positive control)which has an IC50 value of 54.97μmol L^(-1).A mode of action study indicated that kaempferol inhibited viral penetration and replication stages,decreasing viral loads by 4-and 30-fold,respectively.Addition of kaempferol within 16 h post infection(hpi)could significantly inhibit virus replication,and viral genome copies were decreased by almost 15-fold when kaempferol was added at 2 hpi.Kaempferol regulated the NF-κB and MAPKs signaling pathways involved in PRV infection and changed the levels of the target genes of the MAPKs(ATF-2 and c-Jun)and NF-κB(IL-1α,IL-1βand IL-2)signaling pathways.The findings of the current study suggest that kaempferol could be an alternative measure to control PRV infection.

Development and optimization of a double antibody sandwich ELISA for the detection of goose T cell surface CD8α molecule

CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay（DAS-ELISA） for specific detection of goose CD8α（go CD8α）. The results showed that the optimal coated antibody and antigen dilutions were 1：50（the antibody titer was 1：12 800） and 1：32（0.3 ng m L^–1）, respectively, while the optimal capture antibody and horseradish peroxidase（HRP）-labelled goat anti-rabbit Ig G dilutions were 1：50（the antibody titer was 1：51 200） and 1：4 000（the antibody titer was 1：5 000）, respectively. The optimal blocking buffer was 5% bovine serum albumin（BSA）. The best incubating condition was overnight at 4℃, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10^–3 ng m L^–1. Most importantly, go CD8α expression levels in goose spleen mononuclear cells（MNCs） post-Goose parvoviruse（GPV） infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of go CD8α.

The reproductive toxicity of saponins isolated from Cortex Albiziae in female mice

Saponin frsom Cortex Albiziae(SCA) are extensively used in the clinical treatment of tumor and depression. However, SCA may cause several adverse effects, including reproductive toxicity. The present study was designed to assess the mechanism by which SCA cause reproductive toxicity in female mice. The general reproductive toxicity testing was accomplished in female Kunming mice. The animals were divided into four groups: three groups that were treated by oral gavage with 135, 270, and 540 mg·kg-1·d-1 of SCA prepared in physiological saline, respectively, and one vehicle control group that was treated with physiological saline only. The gestational toxicity tests were conducted at 540 mg·kg-1·d-1. The general reproductive toxicity results showed that the pregnancy rate of the SCA-treated group decreased with the pregnancy rate being decreased by 70% at 540 mg·kg-1·d-1. SCA elicited maternal toxicity in the ovary and the uterus, but no fetal toxicity or teratogenicity was observed. The rates of implantation in the early, middle, and late pregnancy were all decreased, with stillbirths and maternal deaths being observed. Histopathological changes showed that SCA adversely affected the ovary and the uterus. In conclusion, SCA-induced reproductive toxicity in female mice is most likely caused by its damage to the ovary and the uterus.