摘要
猪轮状病毒(Porcine Rotavirus,PRV)属于呼肠病毒科轮状病毒属，是引起仔猪病毒性腹泻的主要病原之一，该病在世界范围内分布，给世界各地的畜牧业造成严重的损失。
本研究根据GenBank中已发表的猪轮状病毒VP4全基因序列，设计合成一对引物，以PRV/JL94株RNA为模板，通过RT-PCR技术扩增出约2.3kb的基因片段，命名为VP4。将其与pMD18-T载体连接，构建重组质粒VP4-pMD18-T，对扩增的VP4全基因进行序列测定。测序结果表明JL94-VP4基因全长2362bp，含有一个完整的开放阅读框架，编码776个氨基酸。将测序结果与国外分离株BEN-307株、Gottfried株VP4全基因序列比较，氨基酸同源性分别为96.06%和71.88%。说明JL94株同BEN-307株属于同一VP4血清型，而同Gottfried株属于不同血清型。
研究资料表明PRV VP4基因5’端长750bp的特异性片段主要决定其活性，据此又设计合成一对引物，克隆VP4基因5’端1bp-756bp(包括全部的VP8区、胰酶区和VP5的N端)，命名为VP4主要抗原位点基因；该主要抗原位点基因与PRV国外分离株CRW-8株、Gottfried株该基因片段比较，氨基酸同源性分别为96.43%和67%。同型之间高度保守，不同型间差别较大，其中aa81-aa207变异最大。
将PRV/JL94-VP4主要抗原位点基因同载体pGEX-6P-1连接后转化入E.Coli.BL21 (DE3)plysS，经IPTG诱导表达出50kDa的融合蛋白，与预计相符，对表达产物进行光密度扫描定量分析，融合蛋白占菌体总蛋白的26.6%。对表达的蛋白进行Western blot分析和谷胱甘肽琼脂糖亲和层析纯化，得到纯化的VP4融合蛋白。Western blot证实所表达的融合蛋白有较好的反应原性。
本研究在国内首次克隆了PRV/JL94株VP4全基因，并获得了VP4主要抗原位点基因的表达，为研究我国轮状病毒的病毒毒力的相关基因位点、抗原变异、VP4蛋白的致病机理研究及基因工程疫苗的研制提供依据。
Porcine Rotavirus(PRV) belongs to the family reoviridae. It is one of the major pathogens of diarrhea in piglets and causes of significant losses in graziery worldwide.
According to the reported complete VP4 nucleotide sequences of group A PRV in GenBank，a pair of primers was designed to amplify VP4 gene of JL94. The segments of complete gene 4 of JL94 were obtained from RT-PCR using dsRNA extracted from the virus as template. The product of PCR named VP4 is approximate 2.3kb in length. The VP4 gene was cloned into pMD18-T vector and sequenced. The results of sequencing showed that JL94 isolate complete gene 4 was 2362bp and had a complete open reading frame which encoded 776 amino acides. The sequence was analyzed homologously in comparison with the VP4 nucleotide sequences of two reference porcine rotavirus BEN-307 and Gottfried. The deduced animo acid sequences were 96.06% and 71.88% correspondingly. The result affirmed that JL94 isolate is more similar to BEN-307 than Gottfired in gene type.
According to date specific major antigen site of VP4 is in 5’ extremity 750bp in legs, another pair of primers was designed to amplify VP4 gene of major antigen site(1bp-756bp). The sequence was analyzed homologously in comparison with the VP4 nucleotide sequences of two reference porcine rotavirus CRW-8 and Gottfried. The deduced animo acid sequences were 96.43% and 67% correspondingly. The most divergence of amino acid sequence is located in a region delimited by aa81-aa207.
The major antigen site of VP4 gene was into expression plasmid pGEX-6P-1. The recombinant plasmid VP4-pGEX-6P-1 was transformed into E.coliBL21(DE3)plysS and induced with IPTG. A fusion protein about 50kDa as we expected was found. The amount of the goal protein was evaluated by densitometric scanning. It indicated that the 50kDa product of the VP4 gene was 26.6% of total bacterial protein of BL21. The expressed products were purified by Glutathion-Sepnarose chelation affinity chromatography and tested by western blot. The results showed that GST-VP4 fusion proteins have a positive reaction with anti-JL94 serum.
This study is first time in amplify complete JL94/VP4 gene and get expression of major antigen site of JL94-VP4, it provides the basis evidence and material basis for PRV identification, molecular epdiemiology investigation research of diagnostic reagent and genetic engineering vaccine.
Canditate: Song Yan
Speciality: Preventive Veterinary Science
Supervisor: Prof.Li Yijing
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