【作者】 黄英；

【导师】 张安定；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2020， 硕士

【摘要】 鸡传染性贫血病毒(Chicken infectious anemia virus,CIAV)是一种临床上能够引起雏鸡再生障碍性贫血和全身淋巴组织萎缩为主要特征的鸡传染性贫血(Chicken infectious anemia,CIA)的病原体。CIAV会攻击鸡的免疫系统,不仅会引起其他病原继发感染,还会干扰重大疫病的免疫效果。该病可垂直传播,通过对种鸡进行抗体监测,及时发现并淘汰患病种鸡对于该病的防控至关重要。国外的商品化CIAV ELISA抗体检测试剂盒,价格昂贵,用于该病的抗体检测成本太高,而国内还没有相关的商品化检测产品。因此,本论文以CIAV的衣壳蛋白VP1为抗原,建立了一个特异性、敏感性、重复性较好的CIAV间接ELISA抗体检测方法,希望能够为CIAV的抗体检测和疫病净化提供一个重要工具。主要研究内容如下:1、利用杆状病毒表达系统表达重组VP1蛋白将构建好的含有VP1基因的转移质粒和杆状病毒基因组共转染sf 9细胞,以产生携带有VP1基因的重组杆状病毒,命名为BAC-VP1。通过光学显微镜可以观察到转染细胞相较于正常细胞而言,细胞体积变大,膨胀,初步判断细胞转染后形成了重组杆状病毒。提取感染重组毒的细胞基因组并用鉴定引物进行PCR扩增,PCR扩增产物经核酸电泳检测到相应的目的条带,进一步确定重组杆状病毒BAC-VP1构建成功。收获感染BAC-VP1的细胞后制样,进行Western Blot分析,结果显示,在预测的重组VP1蛋白大小附近,没有检测到相应的条带,说明没有成功表达重组VP1蛋白。2、利用原核表达系统表达重组VP1蛋白进一步用大肠杆菌表达系统实现重组VP1蛋白的表达。根据实验室前期分离到的CIAV基因序列并结合大肠杆菌密码子偏爱性分析,合成了衣壳蛋白VP1的编码序列。分别将截短的重组VP1(40～449aa)蛋白和重组VP1(78～449aa)蛋白表达质粒转化BL21菌株,经IPTG诱导后,SDS-PAGE结果显示成功表达重组VP1(40～449aa)蛋白和重组VP1(78～449aa)蛋白,但是均以不溶性的包涵体形式存在。为了实现重组VP1蛋白的可溶性表达,先后尝试用重组VP2蛋白和5种不同的分子伴侣帮助其正确折叠,最终利用3号分子伴侣(dna K-dan J-grp E)成功实现了重组VP1(40～449aa)蛋白的可溶性表达,利用5号分子伴侣(dna K-dan J-grp E/gro ES-gro EL-tig)成功实现了重组VP1(78～449aa)蛋白的可溶性表达。3、纯化原核表达的重组VP1蛋白利用Ni-NTA亲和层析柱分别对大肠杆菌表达的重组VP1(40～449aa)蛋白和重组VP1(78～449aa)蛋白进行纯化,洗脱后的样品分别用SDS-PAGE分析,结果显示重组VP1(40～449aa)蛋白没有挂上Ni-NTA亲和层析柱,洗脱的样品中没有目的蛋白;而重组VP1(78～449aa)蛋白在300mmol/L咪唑浓度洗脱下,洗脱液中重组VP1蛋白的浓度较高,纯度较好,说明试验成功纯化出重组VP1(78～449aa)蛋白。4、以原核表达的重组VP1蛋白为抗原的CIAV间接ELISA抗体检测方法的建立以纯化后的原核表达重组VP1(78～449aa)蛋白为检测抗原,旨在建立一种CIAV间接ELISA抗体检测方法。通过对抗原包被浓度、血清稀释倍数、包被条件、封闭时间及酶标抗体稀释倍数进行优化,最终确定CIAV间接ELISA抗体检测方法的最佳反应条件:抗原包被浓度为16μg/m L;血清稀释倍数为1:80;包被条件为4℃包被过夜;封闭时间为37℃下封闭2h;酶标抗体稀释倍数为1:10 000。用该方法对30份CIAV阴性血清进行检测,确定该方法的阴阳临界值为0.285。用建立的CIAV间接ELISA抗体检测方法检测AIV、NDV、MDV和IBDV的病原阳性血清时均为阴性,表明该方法的特异性较好;用该方法和IDEXX的CIAV ELISA抗体检测试剂盒同时对不同稀释倍数的CIAV阳性血清进行检测,该方法的最低检测稀释度为1:512,而IDEXX的CIAV ELISA抗体检测试剂盒的最低检测稀释度为1:256,说明该方法的敏感性较好;重复性试验显示批内和批间变异系数均小于10%。应用该方法和IDEXX的CIAV ELISA抗体检测试剂盒同时对90份临床血清进行检测并计算它们之间的符合率,结果显示,IDEXX的CIAV ELISA抗体检测试剂盒检出阳性样本数为50份,其中45份由本方法检出为阳性,阳性样品的符合率为90%;IDEXX的CIAV ELISA抗体检测试剂盒检出阴性样本数为40份,其中32份由本方法检出为阴性,阴性样品的符合率为80%,总符合率为85%。更多还原

【Abstract】 Chicken infectious anemia virus(CIAV)is a pathogen of chicken infectious anemia(CIA),which can cause clinically aplasia bone marrow in young chickens and systemic lymphoid tissue atrophy.CIAV will attack the host’s immune system,which will not only cause secondary infections of other pathogens,but also interfere with the immune effect of major diseases.The disease can be passed from hen to offspring,so it is very important to timely detect and eliminate diseased breeders by monitoring antibody of breeders.Foreign commercial ELISA CIAV antibody detection kits are expensive,and the cost of antibody detection for this disease is too high,there are no related commercial detection products for CIAV in China.Therefore,in this study,the CIAV capsid protein VP1 was used as an antigen to establish a CIAV indirect ELISA antibody detection method with specificity,sensitivity,and good repeatability,hoping to provide an important tool for CIAV antibody detection and disease elimination.The main contents are as follows:1.Expression of recombinant VP1 protein by baculovirus expression systemThe transfer plasmid containing the VP1 gene and the baculovirus genome were co-transfected into sf 9 cells to produce a recombinant baculovirus carrying the VP1 gene,which was named BAC-VP1.Under the light microscope,it was observed that the transfected cells became larger and swelled compared to normal cells,and it was initially determined that the recombinant baculovirus was formed.The genome of cells infected with recombinant virus was extracted and amplified by PCR.The PCR product was subjected to nucleic acid electrophoresis and the target band was detected,it was further confirmed that the recombinant baculovirus BAC-VP1 was successfully constructed.After harvesting the cells infected with BAC-VP1,the samples were subjected to Western Blot.No target band was detected,indicating that the recombinant VP1 protein was not successfully expressed.2.Expression of recombinant VP1 protein by prokaryotic expression systemFurthermore,E.coli expression system was used to realize the expression of recombinant VP1 protein.The CIAV gene sequence isolated in our laboratory was optimized for E.coli codons and then sent to a biological company for synthesis.The truncated recombinant VP1(40～449aa)protein and recombinant VP1(78～449aa)protein expression plasmids were respectively transformed into BL21 strain.After induction by IPTG,the results of SDS-PAGE showed that the recombinant VP1(40～449aa)protein and the recombinant VP1(78～449aa)protein were successfully expressed and were mainly insoluble.Subsequently,recombinant VP2 protein and five different molecular chaperones have been tried to help recombinant VP1 protein achieve soluble expression.Finally,the soluble expression of recombinant VP1(40～449aa)protein was successfully realized by using molecular chaperone No.3(dna K-dan J-grp E),and the soluble expression of recombinant VP1(40～449aa)protein was successfully realized by molecular chaperon No.5(dna K-dan J-grp E/gro ES-gro EL-tig).3.Purification of recombinant VP1 protein expressed in prokaryoticThe truncated recombinant VP1(40～449aa)protein and recombinant VP1(78～449aa)protein expressed in prokaryotic cells were purified by affinity chromatography,and the eluted protein samples were analyzed by SDS-PAGE.The results showed that the truncated recombinant VP1(40～449aa)protein did not bind to the Ni-NTA affinity chromatography column,and there was no target protein in the eluted sample,while the recombinant VP1(78～449aa)protein yielded more and the purity was higher under the concentration of 300 mmol/L imidazole.4.Establishment of CIAV indirect ELISA antibody detection method based on recombinant VP1 protein expressed by E.coli BL21 strainThe purified recombinant VP1(78～449aa)protein expressed by E.coli BL21 strain was used as a diagnostic antigen to establish a CIAV indirect ELISA antibody detection method.The optimal reaction conditions of a CIAV indirect ELISA antibody detection method were determined as follow: the concentration of coating antigen was 16 μg/m L,sample serum was diluted in 1:80,the coated condition was overnight in 4℃,the blocking time was 2h in 37℃,and the enzyme conjugated antibody was diluted in 1:10 000.30 CIAV negative sera were tested by this method,which determined that the value of cut-off was 0.285 in this method.When using the established CIAV indirect ELISA antibody detection method to detect the pathogenic sera of AIV,NDV,MDV,and IBDV,the results are all negative,indicating that the method has good specificity.Using this method and IDEXX’s CIAV ELISA antibody detection kit to simultaneously detect CIAV positive sera of different dilution multiples,the minimum detection dilution of this method is 512-fold dilution,while IDEXX’s CIAV ELISA antibody detection kit is 256-fold dilution,indicating that the method has good sensitivity.Repeatability tests show that the coefficients of variation within and between batches are less than 10%.Using this method and IDEXX’s CIAV ELISA antibody test kit to simultaneously detect 90 clinical sera and calculating their coincidence rate,the results showed that IDEXX’s CIAV ELISA antibody test kit detected 50 positive samples,of which 45 samples were positive by this method and the coincidence rate of positive samples is 90%;IDEXX’s CIAV ELISA antibody test kit detected 40 negative samples,of which 32 samples were negative by this method,and the coincidence rate of negative samples is 80%,the overall coincidence rate is 85%.更多还原