猪繁殖与呼吸综合征病毒HuN4-F112弱毒株NSP2区表达猪瘟病毒E2基因表位的研究

Study on Using NSP2 Protein of Porcine Reproductive and Respiratory Syndrome Virus(HuN4-F112) to Express E2 Neutralizing Epitope of Classical Swine Fever Virus

建立共表达猪瘟病毒(Classic swine fever virus,CSFV)E2蛋白主要中和性抗原表位基因的猪繁殖与呼吸综合征病毒(PRRSV)反向遗传操作平台,为进一步研究PRRSV作为病毒载体提供实验数据。本研究在PRRSVHuN4-F112疫苗株感染性分子克隆的基础上,采用突变PCR技术将CSFV E2蛋白的主要中和性抗原表位基因插入HuN4-F112弱毒株PRRSV已鉴定出的两个NSP2蛋白的复制非必需区,经基因片段的克隆、拼接,构建了两株含有CSFV E2蛋白的主要中和性抗原表位基因的感染性PRRSV cDNA克隆psk-HuN4-F112-Δ508-532+E2和psk-HuN4-F112-Δ480-532+E2。用限制性内切酶SwaΙ分别将两株共表达的感染性克隆线性化,之后通过细胞外转录获得病毒RNA,用脂质体法分别将病毒RNA转染BHK-21细胞包装出病毒粒子,再分别将其转接到MARC-145细胞传代拯救出两株病毒。分别对两株拯救病毒进行RT-PCR扩增、酶切和序列分析鉴定。结果表明,两株拯救病毒含有不同于亲本病毒的分子标记(拯救病毒基因组的14 667位因同义突变产生的MluⅠ酶切位点)和插入基因序列。间接免疫荧光试验表明,CSFV E2蛋白主要中和性抗原表位基因均能在两株拯救的PRRSV中表达,且能够在其传代过程中稳定遗传。病毒生长特性结果比较显示两株拯救病毒与亲本病毒在MARC-145细胞上具有相似的增殖特性。本研究构建了两株共表达CSFV E2蛋白主要中和性抗原表位基因的PRRSV感染性克隆并获得了拯救病毒,且外源基因能在拯救病毒中稳定表达,为进一步开发新型PRRSV二联疫苗提供了有效的反向遗传操作平台。

Establishment of recombinant porcine with co-expression E2 Epitope of Classical Swine reproductive and respiratory syndrome virus （PRRSV） Fever virus （CSFV） is a crucial step to develop a genetic engineered vaccine against PRRSV and CSFV. Reverse genetic manipulation could be adopted as a com- monly used technique. In this study, we focus on using nonessential regions of NSP2 （aa480-532 and aa508-532） as viral vector to express E2 Epitope of CSFV. A neutralizing epitope of classical swine fever virus （CSFV） E2 protein was inserted into the two nonessential region of nsp2 by the method of mutant PCR, basing on the infectious clone of HuN4-Fll2 vaccine strain. The co-expressed full-length eDNA clones （psk-HuN4-Fl12-A508-532 q- E2 and psk-HuN4-F112-A480-532 q- E2） were assembled by cloning and splice of the gene fragments. The completely assembled full-length eDNA clones were confirmed by sequence and Swa I enzyme digestion. Capped RNAs were transcribed in vitro from a full-length eDNA clone of the viral genome and transfected into BHK-21cells by liposome to acquire the rescued virus. The rescued recombinant viruses were passaged on MARC-145 ceils. The successfully rescued viruses were tested by RT-PCR, digestion, and genome sequence. The results showed that＇these rescued viruses could be distinguished from the parental virus （HuN4-F112） with the mutant genetic marker （Mlu I enzyme site of virual genome at 14 667nt was created by synonymous mutation） and the inserted nsp2 gene region. The results of IFA showed that the inserted E2 epitope could be expressed by the recombinant viruses and the E2 epitope gene was stable during the viral serial passage. The results of plaque assay and viral growth curve showed that the recovery viruses possessed similar characterses of viral growth to those of the paren- tal virus. In summary, the full-length infectious eDNA clones containing the marker gene were constructed and the marker recombinant viruses were rescued. The results suggested that these stable infectious clones could be used as an important tool for development of novel vaccine against PRRSV.