Significant association between IL-18 and OCT4 gene polymorphisms in susceptibility and clinical characteristics of prostate cancer

Objective Recent studies have shown abnormal expression of octamer-binding transcription factor 4(OCT4) and interleukin-18(IL-18) to be related to cancer. However, the molecular mechanisms by which the IL-18 and OCT4 gene polymorphisms are associated with prostate cancer remain unclear. In this study, we aimed to determine whether the presence of IL-18 and OCT4 polymorphisms were associated with size, grade, tumor, nodes and metastasis(TNM) stage, or survival in patients with prostate cancer.Methods Polymorphisms in OCT4 and IL-18 genes were evaluated to determine susceptibility to prostate cancer in 120 patients. A control group consisted of 125 Chinese participants. Genotyping was performed using TaqMan allelic discrimination assays, and statistical analysis was performed using SPSS. Results No association was found between OCT4 and IL-18 gene polymorphisms and prostate cancer susceptibility. For OCT4 AA and IL-18-607 CC genotypes, there was a significant association with higher tumor grade(P = 0.03 and P = 0.025) and stage(P = 0.04 and P = 0.001). The OCT4 and IL-18-137 GG genotype was correlated with higher tumor grade(P = 0.028) and stage(P = 0.008). Furthermore, OCT4 AA was significantly more frequent in patients with lymph node metastasis(P = 0.02) and distant metastasis(P = 0.01). The Cox proportional hazard model showed that tumor grade and stage grouping were independent prognostic factors but IL-18 and OCT4 polymorphisms were not. Conclusion The OCT4 gene may have a profound effect on prostate cancer risk. Polymorphism variants in the IL-18(IL-18-607 and IL-18-137) and OCT4 genes may be associated with poor prognoses for individuals with prostate cancer.

Single Nucleotide Polymorphisms rs2227284, rs2243283 and rs2243288 in the IL-4 Gene show no Association with Susceptibility to Chronic Hepatitis B in a Chinese Han Population

Objective To investigate the relationship between single nucleotide polymorphisms(SNPs) of the interleukin-4(IL-4) gene and outcome of hepatitis B virus(HBV) infection in a Chinese Han population.Methods Total of 501 patients with chronic hepatitis B virus(HBV) infection and 301 controls with selflimiting HBV infection were studied. Three tag SNPs in the IL-4 gene(rs2227284G/T, rs2243283C/G and rs2243288A/G) were genotyped by the Multiplex snapshot technique. The genotype and allele frequencies were calculated and analyzed.Results The three SNPs showed no significant genotype/allele associations with chronic HBV infection. Overall allele P values were: rs2227284, P = 0.655, odds ratio(OR) [95% confidence interval(CI)] = 1.070(0.793-1.445); rs2243283, P = 0.849, OR(95% CI) = 0.976(0.758-1.257); rs2243288, P = 0.659, OR(95% CI) = 1.060(0.818-1.375). Overall genotype P values were: rs2227284, P = 0.771; rs2243283, P = 0.571; rs2243288, P = 0.902. There were no statistically significant differences between patients with chronic HBV infection and controls. Haplotypes generated by these three SNPs also had no significant differences between the two groups. Conclusions The three tag SNPs of IL-4 were not associated with the outcome of HBV infection in the Han Chinese population.

IBDV VP2/4/3-ChIL-2融合基因真核表达载体的构建及其在Vero细胞中的表达

构建含有传染性法氏囊病病毒(InfectiousBursalDiseaseVirus,IBDV)VP2/4/3和鸡白细胞介素2(ChickenInterleukin2,ChIL2)融合基因的真核表达载体pCIVP2/4/3IL2、pCIIL2VP2/4/3,并在Vero细胞中进行表达.应用重叠延伸剪切技术(splicingbyoverlappingextension),分别经3次PCR获得融合基因片段VP2/4/3IL2、IL2VP2/4/3,定向插入真核表达载体pCI中,获得重组质粒pCIVP2/4/3IL2、pCIIL2VP2/4/3,在脂质体介导下,分别转染Vero细胞.RTPCR检测证实导入的外源基因在Vero细胞中得到了转录;间接免疫荧光试验(IFA)和Westernblot检测证实重组质粒在Vero细胞中正确表达了插入的外源基因编码的融合蛋白,且表达的融合蛋白具有免疫反应性.重组真核表达载体pCIVP2/4/3IL2、pCIIL2VP2/4/3的成功构建及其在Vero细胞中的有效表达,为进一步探讨ChIL2作为分子免疫佐剂对IBDVDNA疫苗的特异性免疫增强作用奠定了基础.

鸡IL-2和IL-4融合基因真核表达质粒的构建及其表达产物的生物学活性

为提高鸡球虫病活疫苗的免疫效果并降低其副作用,本试验用合成的鸡白介素2(chIL-2)和鸡白介素4(chIL-4)基因片段构建3个融合基因真核表达质粒,并利用脂质体法将其转染至原代鸡胚盲肠上皮细胞,通过实时荧光定量PCR(RT-qPCR)检测其表达水平,MTT法测定其表达的分泌产物活性。结果显示:成功构建的pCI-chIL-4-chIL-2-EGFP、pCI-chIL-2-EGFP、pCI-chIL-4-EGFP重组真核表达质粒能在原代鸡胚盲肠上皮细胞中高效表达;3种重组真核表达质粒转染后24~96 h的鸡脾淋巴细胞活性极显著高于空载组(P<0.01),且在72 h时达峰值;而单一因子组鸡脾淋巴细胞增殖活性极显著低于pCI-chIL-4-chIL-2-EGFP组(P<0.01)。由此可见,本试验成功构建了能表达分泌chIL-2和chIL-4的融合基因重组质粒;且chIL-4-chIL-2融合蛋白生物学活性显著高于单一因子。

Impact of Yupingfengsan Oral Liquid on the Expression of IL-4, IFN-<i>γ</i>and NF-<i>κ</i>B of Allergic Rhinitis Mice

To observe the impact of Yupingfengsan Oral Liquid on the expression of IL-4 and IFN-γ in AR mice’s serum and expression level of nuclear factor—κB (NF-κB) protein and gene in nasal mucosa. Method: Forty BALB/c mice were randomly divided into the normal group, model group, Yupingfengsan Oral Liquid group (6 g/kg) and Loratadine group, with 10 mice per group. AR mice model was established by OVA, and IL-4 and IFN-γ contents can be measured with ELISA. The morphological changes of nasal mucosa were observed by hematoxylin eosin (HE) staining and NF-κB expression in the nasal mucosa of mice was tested with Real-Time PCR and Western blot. Results: Compared with the model group, the nasal symptoms in the Yupingfengsan Oral Liquid group and Loratadine group were obviously relieved. HE staining showed that there was a little inflammatory cell infiltration in the nasal mucosa of Yupingfengsan Oral Liquid group and Loratadine group and it was significantly reduced when compared with the model group. IL-4 level in the serum and expression of NF-κB protein and gene in the nasal mucosa was consistent and it was decreased when compared with the model group (P < 0.01), but the IFN-γ level in the serum was increased (P < 0.01). Conclusion: Yupingfengsan Oral Liquid can improve the clinical symptoms and histopathological manifestations of AR mice sensitized by OVA, inhibit the NF-κB expression, balance the percentage of Th1/Th2 cells, increase the IFN-γ level in the serum and decrease the IL-4 level.

Expression of toll-like receptor 4 gene polymorphism and inflammatory factors in peripheral blood on patients with sepsis

Objective:To observe the presence of toll-like receptor 4(TLR4)gene polymorphism in the venous blood in patients with sepsis,meanwhile,to observe the expression and the diagnostic value of TLR4 mRNA,interferon-(IFN-),interleukin-23(IL-23),procalcitonin(PCT)and C-reactive protein(CRP)in patients with different degrees of sepsis.Methods:The peripheral blood samples from the subjects were collected to extract genomic DNA,gene sequencing and enzyme digestion method were applied to the detection of TLR4 Asp299Gly loci polymorphism.The expression of TLR4 mRNA in the peripheral blood was detected by reverse transcriptase polymerase chain reaction(RT-PCR).The expression levels of IFN-,IL-23,PCT and CRP were measured by enzyme linked immunosorbent assay(ELISA).Results:(1)The gene polymorphism was not present in TLR4 Asp299Gly loci;(2)The expression of TLR4 mRNA in the peripheral blood in patients with sepsis:on d1,there were statistically significant differences between the normal group and the group(APACHE II≤20),between the normal group and the group(APACHE II>20),between the group(APACHE II≤20)and the group(AAPACHE II>20)(t=5.741,14.780 and 10.500,all p<.01).APACHE II stands for Acute Physiology and Chronic Health Evaluation II.On d7,there were statistically significant differences between the normal group and the group(APACHE II≤20),between the normal group and the group(APACHE II>20),between the group(APACHE II≤20)and the group(APACHE II>20)(t=4.186,13.830 and 9.645,all p<.01);(3)The expression levels of IFN-,IL-23,PCT and CRP were obviously upregulated(all p<.01),and TLR4 was positively correlated with IFN-and IL-23(all p<.01);(4)The best cutoff value of TLR4 mRNA at baseline was 891.6μg/L,with a sensitivity of 100%and a diagnostic specificity of 57%.The best cutoff value of IFN-at baseline was 84.5μg/L,with a sensitivity of 100%and a diagnostic specificity of 57%.The best cutoff value of IL-23 at baseline was 861μg/L,with a sensitivity of 100%and a diagnostic specificity of 97%.Conclusions:(1)The Asp299Gly polymorphism is not present in TLR4 gene;(2)The expression levels of TLR4 mRNA,IFN-and IL-23 are relatively high in patients with sepsis,and will be higher with the increased severity of sepsis;(3)TLR4 mRNA,IFN-and IL-23 can be used as molecular biomarkers for the early diagnosis of sepsis.

鸡白细胞介素4基因的克隆及其原核表达

应用RT-PCR技术,从被诱导的鸡脾脏淋巴细胞中扩增鸡白细胞介素-4(ChIL-4)基因cDNA,经克隆筛选和测序后,构建出重组质粒pGEX-6P-ChIL-4和pET-ChIL-4,分别转化相应的受体菌,经IPTG诱导和SDS-PAGE分析,结果表明ChIL-4基因在上述载体中均获得表达,融合蛋白大小分别为38ku和18ku,Westernblot结果也显示,在相对分子质量38ku和18ku位置有特异性条带。这为重组ChIL-4的规模化生产、疫苗佐剂的研制及其单克隆抗体的制备奠定了基础。

广西黄鸡β-防御素Gal-4基因的克隆和体内诱导表达

采用RT-PCR方法从广西黄鸡骨髓中扩增了β-防御素Gal-4基因的cDNA片段。序列分析结果表明,获得的广西黄鸡Gal-4基因大小为321 bp,推导的Gal-4由63个氨基酸组成,其中N端20个氨基酸为信号肽,C端38个氨基酸组成Gal-4的成熟肽。另外,分析了Gal-4基因在正常广西黄鸡(对照组)和H9N2禽流感病毒感染的广西黄鸡(感染组)的肺、肝、腔上囊、十二指肠、肾、气管组织中的表达情况。分析结果表明,在检测的6个组织中,肺、十二指肠、肾组织Gal-4基因的表达在感染组和对照组之间没有明显的差别,而在肝、气管和腔上囊组织中的表达有明显差异,感染组比对照组的Gal-4阳性样品数多。在肝组织中,对照组的30个样品检测到26个阳性样品,而感染组的30个样品全都是阳性；在气管组织中,对照组30个样品检测到19个阳性样品,而感染组的30个样品检测到24个阳性样品；在腔上囊组织中,Gal-4基因的诱导表达情况非常明显,对照组30个样品中仅有9个阳性,而感染组的30个样品中有21个阳性。

鸡黑素皮质素受体-4反向激动剂细胞筛选模型的建立

本试验旨在建立体外鸡黑素皮质素受体-4(cMC4R)反向激动剂的细胞筛选模型,为从中草药中筛选出能与cMC4R作用且存在活性的中药成分提供一些基础性的研究。将成功构建的cMC4R真核表达质粒cMC4RpcDNA3.1(+)-myc与报告基因质粒pGL4.29[luc2p/CRE/Hygro]按1∶5的比例共转染到CHO-K1细胞,两周后经有限稀释法获得单克隆;将获得的单克隆分别用CRE激活剂Forskolin和MC4R激动剂NDP-MSH作用6h,挑选萤火虫荧光素酶的相对表达量最高的单克隆作为阳性细胞株;提取阳性细胞总RNA,RT-PCR检测阳性细胞内cMC4R基因、荧光素酶报告基因的转录水平;对MC4R激动剂NDP-MSH的终浓度、NDP-MSH孵育时间及溶剂DMSO的终浓度进行优化,应用Z’因子对筛选方法进行评估。结果显示,获得了5个单克隆(A2、F7、F8、F9、H10),其中单克隆A2的萤火虫荧光素酶的相对表达量最高,将其作为阳性细胞株;RT-PCR在预期位置扩增出大小分别为996和1 653bp的两条目的条带;激动剂NDP-MSH终浓度为10-9 mol/L、NDP-MSH孵育时间为8h、溶剂DMSO终浓度小于2%时,筛选模型Z’因子为0.82。结果表明,本试验所建立的筛选方法可靠、稳定,细胞筛选模型可用于cMC4R反向激动剂的筛选,为进一步从中药中筛选出有效物质奠定了基础。

孟鲁司特联合沙美特罗替卡松对咳嗽变异性哮喘患者白介素-4和γ-干扰素水平的影响

目的探讨孟鲁司特联合沙美特罗替卡松对咳嗽变异性哮喘患者白介素-4(IL-4)和γ-干扰素(IFN-γ)水平的影响。方法选择我院收治的咳嗽变异性哮喘患者74例,随机分为实验组和对照组。两组患者均予以沙美特罗替卡松吸入,1吸/次,2次/d。实验组在此基础上口服孟鲁司特咀嚼片10 mg,1次/d,连用12周。观察两组患者治疗前后血清IL-4和IFN-γ水平的变化,并观察临床疗效及安全性。结果治疗12周后,两组患者血清IL-4水平较治疗前明显下降,IFN-γ水平较治疗前明显上升(P<0.01或P<0.05),且实验组下降或上升值明显大于对照组(P<0.05);同时实验组的临床总有效率明显高于对照组(χ2=4.16,P<0.05)。对照组和实验组治疗过程中分别出现不良反应3例和6例,症状较轻,未发生严重药物不良反应。两组治疗过程中不良反应发生率比较差异无统计学意义(χ2=0.51,P>0.05)。结论孟鲁司特联合沙美特罗替卡松治疗咳嗽变异性哮喘效果明确,能降低血清IL-4水平,提高血清IFN-γ水平,纠正Thl/Th2细胞因子平衡失调,且安全性较高。

禽腺病毒血清4型感染鸡组织中NLRP3基因转录水平的实时荧光定量PCR分析

旨在分析禽腺病毒血清4型(FAdV-4)感染鸡组织中NLRP3基因的转录水平,本研究设计鸡NLRP3特异性引物,利用RT-PCR扩增NLRP3基因180 bp片段并克隆至pMD-18T载体,制备重组质粒pMD-18T-NLRP3。以pMD-18T-NLRP3质粒作为标准品进行荧光定量PCR并建立标准曲线。通过反应条件优化,成功建立了检测NLRP3基因的实时荧光定量PCR方法,并利用该方法对致病性FAdV-4感染鸡组织中NLRP3基因的转录水平进行了分析。结果显示,所设计的NLRP3引物可特异性扩增鸡NLRP3基因,建立的实时荧光定量PCR对鸡NLRP3标准质粒的扩增曲线良好,标准品的拷贝数与Cq值呈现良好的线性关系。与对照组相比,NLRP3分子在FAdV-4感染鸡肝和脾中的转录水平极显著高于对照组(P<0.001),在盲肠扁桃体和法氏囊的表达显著高于对照组(P<0.01)。本研究所建立的鸡NLRP3基因SYBR GreenⅠ实时荧光定量PCR可以检测FAdV-4感染鸡不同组织中NLRP3的转录水平;致病性FAdV-4感染所造成的组织炎症损伤与NLRP3分子密切相关。

Gene polymorphisms of interleukin-28, p21-activated protein kinases 4, and response to interferon-α based therapy in Chinese patients with chronic hepatitis B

Background Peg-lnterferon-a treatment is expensive and associated with considerable adverse effects, selection of patients with the highest probability of response is essential for clinical practice. The objective of this study was to assess the relationship between the gene polymorphisms of interleukin-28 （IL-28）, p21-activated protein kinase 4 （PAK4） and the response to interferon treatment in chronic hepatitis B patients. Methods Two hundred and forty interferon-naive treatment HBeAg seropositive chronic hepatitis B patients were enrolled in the present prospective nested case-control study. Peripheral blood samples were collected, including 92 with favorable response and 148 without response to the interferon treatment. Rs8099917, rs12980602, and rs9676717 SNP was genotyped using matrix assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF MS）. Results IL-28 genotype was not associated with response to interferon treatment （OR for GT/GG vs. TT, 0.881 （95% CI 0.388-2.002）; P=0.762; OR for CT/CC vs. TT, 0.902 （95% CI 0.458-1.778）; P=-0.766）. Rs9676717 in PAK4 genotype was independently associated with the response （OR for CT/CC vs. TT, 0.524 （95% CI 0.310-0.888）; P=0.016）. When adjusting for age, gender, smoking, drinking, levels of hepatitis B virus DNA, and alanine aminotransferase （ALT）, rs9676717 genotype TT appeared to be associated with a higher probability of response for interferon treatment （OR, 0.155 （95% CI 0.034-0.700）; P=0.015）. Conclusion Genotype TTfor rs9676717 in PAK4 gene and no drinking may be predictive of the interferon-a treatment success.

Association between the polymorphisms of interleukin-4, the interleukin-4 receptor gene and asthma

Background Interleukin-4 （IL4） is one of the most important cytokines involved in a variety of allergic disorders, particularly, asthma. A number of genetic epidemiological studies have identified an association between the gene polymorphisms of IL4 and interleukin-4 receptor （IL4R） and asthma in different populations. However, these studies have been inconsistent and inconclusive. The aim of this study was to investigate the association between the single nucleotide polymorphism （SNP） of IL-4, IL-4R and asthma risk in case-controlled studies using meta-analysis. Method A genetic model-free approach was used to perform the meta-analysis. Asthma （atopy status nondefined）, nonatopic and atopic asthma subgroups were separately analyzed. Next, the ethnic subgroup was analyzed. Heterogeneity and publication bias were also explored. Results Only two polymorphisms of IL4 （rs2243250 and rs2070874） and four polymorphisms of IL4R （rs1801275, rs1805011, rs1805010, and rs1805015） were included in the meta-analysis. Polymorphisms rs2243250 and rs2070874 of IL-4 and rs1801275 and rs1805011 of IL4R were associated with asthma. The overall odds ratio （OR） of rs2243250 in the CC versus TT＋TC genotypes was 0.84 （95% CI： 0.75-0.94）, and the Z-test for the overall effect was 3.0 （P=0.003）. We obtained significant results from this polymorphism in the Caucasian ethnicity and adult groups. However, the overall OR of rs1801275 for the GG＋AG versus AA genotype was 1.16 （95% CI： 1.00-1.35）, and the Z-test for the overall effect was 1.87 （P=0.06）. Moreover, significant results were only obtained from the sub-group analysis in Asians （P=0.02）. In the rs1805011 polymorphism of IL4R, the overall OR for the CC ＋AC versus AA genotypes was 0.39 （95% CI： 0.16-0.95）, and the Z-test for the overall effect was 2.08 （P=0.04）. Conclusions Both the IL4 and IL4R polymorphisms were associated with asthma. The rs2243250 polymorphism of IL4 was more important in the white and adult groups. Individuals who carried the C allele for rs2070874 of the IL4 gene demonstrated increased asthma risk compared to TT homozygotes. An individual with an AA genotype in rs1805011 of the IL4R gene was less likely to suffer from asthma compared to the other two genotypes.

Immune Responses to Trichloroethylene and Skin Gene Expression Profiles in Sprague Dawley Rats

Objective To characterize the immune reaction in SD rats exposed to trichloroethylene （TCE） and to identify the gene expression profiles involved in skin after TCE exposure. Methods Fifteen percent of TCE was injected intradermally into the rat back （100 μL/120 g） at intervals of 7 days. Whole blood was collected 24 h after the fifth or seventh intradermic administration of TCE. The percentages of CD4＋ and CD8＋ of T lymphocytes were measured by a flow cytometer. The concentrations of IFN-gamma and 1L-4 in the serum were semi-quantified by ELISA. Total RNAs of skin samples at 3 h or 24 h after the seventh dose of TCE in SD rats were extracted, and gene expression proftles of these tissues were analyszed by rat toxicology U34 array of Affymetrix. Results Obvious decline of CD4＋ in T lymphocytes was observed in the TCE-administer group. No significant concentration differences in IFN-gamma and IL-4 were found between TCE-treated and control rats. Gadd45a and Mel were significantly up regulated in skin tissue 24 h after TCE exposure. The expression regulation of immune response factors was as active as proteins associated with lipid metabolism and synthesis process in these skin samples of SD rats exposed to TCE. Conclusion T-helper type 1 cells mediate immune response can not be elicited in TCE-treated SD rats, but certain immune disorder can be induced.

鸡外周血T淋巴细胞分群及免疫相关基因表达分析

为探索鸡抗病育种中简便实用的免疫功能测定指标,试验采用成像流式细胞术对四川山地乌骨鸡和大恒699肉鸡配套系的4周龄和10周龄外周血进行T淋巴细胞亚群分型,并检测外周血中白介素6(IL-6)、Toll样受体3(TLR3)和干扰素(IFN-γ)基因的表达水平。结果显示:10周龄时,CD_(4)^(+)T淋巴细胞亚群在两鸡种间差异显著(P<0.05),4周龄和10周龄的两鸡种CD_(8)^(+)T淋巴细胞亚群均有极显著差异(P<0.01);CD_(4)^(+)/CD_(8)^(+)比值在0.7~1.6之间呈正态分布,且随周龄增加而降低;大恒699肉鸡配套系的CD_(4)^(+)/CD_(8)^(+)比值比四川山地乌骨鸡高;IL-6和IFN-γ表达量随周龄增加而增加,但TLR3相反。细胞因子的增多和CD_(4)^(+)/CD_(8)^(+)比值的降低表明鸡只随周龄增长,机体免疫平衡发生改变。结果表明,基于成像流式细胞术进行鸡外周血T淋巴细胞分型简易可行,CD_(4)^(+)/CD_(8)^(+)比值可作为鸡免疫力检测的一个指标;天然免疫相关基因IL-6、TLR3和IFN-γ基因的表达量可以作为免疫力检测的参考。

鸡IL-4和IL-2重组融合基因佐剂对鸡球虫活疫苗的增效作用研究

为了研究鸡白细胞介素4(IL-4)和IL-2重组基因佐剂pCI-chIL-4-chIL-2-EGFP对鸡球虫活疫苗的增效作用,试验采用基因佐剂配合鸡球虫活疫苗对雏鸡进行免疫攻毒试验,测定该基因佐剂的最小有效剂量、给药途径,使用该基因佐剂对鸡球虫活疫苗的最短免疫间隔时间和免疫产生期的影响。结果表明:抗球虫指数(ACI)和相对增重率(RWG)随着基因佐剂剂量增高而升高,肌肉注射100μg/只时,ACI(189.32)和RWG(90.35%)达到高峰;口服基因佐剂300μg/只组的ACI(188.41)与肌肉注射基因佐剂100μg/只组接近;肌肉注射佐剂免疫攻毒组的两次免疫最短间隔时间(6 d)较不加基因佐剂组(7 d)缩短1 d;肌肉注射佐剂免疫攻毒组的免疫产生期为二免后第5天,较无基因佐剂免疫攻毒组提前1 d。说明肌肉注射IL-4和IL-2重组基因佐剂pCI-chIL-4-chIL-2-EGFP 100μg/只可提高鸡球虫活疫苗的免疫效果,缩短免疫产生期和免疫间隔。

Retrovirus-mediated delivery of an IL-4 receptor antagonist inhibits allergic responses in a murine model of asthma

This work reports the investigation of the effect of airway IL-4RA gene transfer by a recombinant retroviral vector on airway inflammation and airway responsiveness in asthmatic mice. The retrovirus-mediated delivery of IL-4RA to the airways of mice inhibited elevations of airway responsiveness and the development of allergic inflammation in asthmatic mice, and regulated the Th1/Th2 balance in OVA-sensitized and -challenged mouse models. This suggests that gene therapy is a therapeutic option for treating and controlling chronic airway inflammation and asthma symptoms.

2017年黑龙江省禽腺病毒4型分离株Hexon基因的遗传特征分析

目的分析2017年黑龙江省禽腺病毒4型(fowl adenovirus-4,FAd V-4)分离株Hexon基因的遗传特征。方法提取疑似心包积液综合征(hydropericardium syndrome,HPS)的病鸡肝脏DNA,以其为模板,经PCR扩增Hexon基因,并进行测序。采用BLAST软件对Hexon基因序列进行数据库比对;采用Meg Align软件进行核苷酸和氨基酸同源性比对;采用Mega 6软件将分离获得的FAd V-4菌株及35个参考FAd V菌株的全长Hexon基因构建系统进化树。结果 Hexon基因PCR产物长度为1 204 bp,将分离株命名为FAd V-4-DQ。与其他8个FAd V分离株核苷酸和氨基酸的同源性分别为34.6%~98.7%和22.0%~95.7%,其中与中国2015年FAd V-4 HNHB分离株及2016年FAd V-4 HN151025分离株的核苷酸和氨基酸的同源性较高,分别为98.7%、95.7%和98.6%、95.2%;其次是与印度2008年的FAd V-4 PJ-06分离株的核苷酸和氨基酸的同源性较高,分别为98.2%和94.4%。基因进化树分析显示,FAd V-4-DQ株与中国2015及2016年的15个分离株基因型属于同一个进化分枝,并由同一祖先进化。结论本研究获得的FAd V分离株属血清型FAd V-4型,FAd V-C种,与中国2015及2016年流行毒株同属一个基因簇,并推测该分离株起源于印度早期菌株。

鸡碳酸酐酶4基因在毕赤酵母中的表达

[目的]通过毕赤酵母的表达获得鸡碳酸酐酶4(CAⅣ)蛋白。[方法]根据鸡CAⅣ的序列,结合毕赤酵母密码子的偏好性,合成CAⅣ基因。将CAⅣ基因克隆到pPICZαA真核表达载体,获得重组表达质粒pPICZαA-CAⅣ。将其电转毕赤酵母GS115后,获得重组毕赤酵母菌GS115/pPICZαA-CAⅣ。用终浓度为1%的甲醇对重组阳性菌进行诱导表达,通过SDS-PAGE和Westernblot法检测蛋白的表达,并用Ni离子亲和层析法对表达出的蛋白进行纯化。[结果]成功构建了表达载体pPICZαA-CAⅣ,转化重组酵母菌后可分泌出36kDa左右的CAⅣ蛋白,并通过Ni离子亲和层析法获得了单一性的目的蛋白CAⅣ。[结论]获得分子量约36kDa的鸡CAⅣ蛋白。

慢性荨麻疹患者户尘螨特异性IgE与IL－4的表达及意义

目的：研究及探讨户尘螨特异性IgE与IL－4水平在慢性荨麻疹病人血清中的表达及相关性。方法：选择对户尘螨过敏的慢性荨麻疹患者41例，健康对照组20例；采用免疫印迹法测定慢性荨麻疹患者血清中户尘螨特异性IgE指标，采用ELISA法测定慢性荨麻疹患者血清中IL－4的指标。结果：慢性荨麻疹病人血清中户尘螨sIgE水平明显高于健康对照组，IL－4指标亦明显高于健康对照组，组间差异有统计学意义（ P＜0．05）；慢性荨麻疹患者血清中户尘螨sIgE与IL－4水平无显著相关性（r为0．171，P＞0．05）。结论：户尘螨特异性IgE、IL－4水平与CU的发病有关；慢性荨麻疹病人血清中IgE水平的影响因素众多。