犬瘟热病毒H基因原核表达及间接ELISA的建立

发布时间：2018-06-15 20:34

本文选题：犬瘟热病毒 + H基因　；参考：《江西农业大学》2015年硕士论文

【摘要】：犬瘟热病毒(canine distemper virus,CDV)是犬瘟热(canine distemper,CD)的病原体。CD是威胁犬类健康的最严重传染病之一,发病后死亡率极高,且无特异性治疗药物。为此,本试验针对CDV的N基因,建立了快速诊断RT-PCR方法;同时通过RT-PCR方法扩增CDV部分H基因,通过原核表达、复性,成功表达出CDV H蛋白,并构建了H蛋白间接ELISA方法,证实表达的H蛋白具有免疫原性,具体内容如下:1.根据GenBank公布的CDV N蛋白基因序列,设计了一对特异性引物,成功扩增出464bp的CDV N基因片段。测序结果表明该序列与GenBank已经发表CDV基因序列的同源性为94.8%~98.5%。利用本文建立的CDV检测RT-PCR方法对30例疑似CDV病例检测显示:RT-PCR检测方法的阳性检出率为90%、胶体金试纸阳性检出率为86.7%,说明RT-PCR方法检出率和敏感性更优。2.血凝素蛋白是诱导机体产生中和抗体的主要蛋白之一。本文去除H基因高度疏水区核苷酸设计引物,成功克隆了来自南昌CDV病料中CDV长度为1236nt的H基因片段。将该基因片段插入到载体pET32a中,构建了原核表达质粒pET32a-CDVH,重组蛋白H基因在Rosetta菌中获得了高效表达。表达产物以包涵体的形式存在,经包涵体洗涤、复性、Ni柱纯化、透析浓缩,最终获得浓度为0.561mg/m L重组H蛋白溶液。小鼠免疫试验显示,重组蛋白免疫产生的血清抗体效价在1:102400以上,免疫原性良好。3.以7.5ug/m L的复性H蛋白溶液包被96孔酶标板,成功建立了重组CDV H蛋白间接ELISA。间接ELISA条件为:重组蛋白包被浓度7.5ug/m L、1%明胶封闭、血清稀释倍数为1:80、酶标二抗稀释倍数为1:5000,阴阳性血清判定的临界值(OD492)为0.100。试验证明该间接ELISA方法特异性强,重复性良好。对收集的背景清楚血样的检测结果符合预期。
[Abstract]:Canine distemper virus (CDV) is the pathogen of canine distemper CD.CD is one of the most serious infectious diseases that threaten the health of dogs. Therefore, a rapid diagnostic RT-PCR method was established for the N gene of CDV, and the partial H gene of CDV was amplified by RT-PCR, and the CDV H protein was successfully expressed by prokaryotic expression and renaturation, and an indirect Elisa method of H protein was constructed. The expressed H protein has immunogenicity, as follows: 1. According to the sequence of CDVN protein gene published in GenBank, a pair of specific primers were designed to amplify the CDVN gene fragment of 464bp successfully. The sequencing results showed that the homology between this sequence and the published CDV gene sequence in GenBank was 94.898. 5%. Using the CDV detection RT-PCR method established in this paper, 30 suspected cases of CDV were detected by RT-PCR. The results showed that the positive detection rate of the two methods was 90 and 86.7 respectively, which indicated that the detection rate and sensitivity of RT-PCR was better than that of gold test paper. Hemagglutinin protein is one of the main proteins that induce the production of neutralizing antibodies. In this paper, the H gene fragment with length of 1236nt from Nanchang disease material was cloned successfully by removing the high hydrophobic nucleotide design primers of H gene. The gene fragment was inserted into the vector pET32a and the prokaryotic expression plasmid pET32a-CDVHwas constructed. The recombinant protein H gene was highly expressed in Rosetta bacteria. The expressed product existed in the form of inclusion body, washed by inclusion body, purified by renaturation Ni column, and concentrated by dialysis. Finally, the recombinant H protein solution with 0.561mg/m L concentration was obtained. The antibody titer of the recombinant protein was above 1: 102400, and the immunogenicity was good. 3. Indirect ELISAs of recombinant 7.5ug/m H protein were successfully established by coating 96 well enzyme labeled plates with H protein solution of 7.5ug/m L renaturation. The indirect Elisa conditions were as follows: the concentration of recombinant protein was blocked by 7.5ug/m 1% gelatin, the dilution multiple of serum was 1: 80, the dilution multiple of enzyme labeled second antibody was 1: 5 000, and the critical value of yin-yang serum was 0.100. The indirect Elisa method was proved to be specific and reproducible. The results of blood samples with clear background were in line with expectations.
【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

【相似文献】