鸡新城疫病毒HeB02分离株F基因的克隆及其DNA疫苗的研究

根据GenBank报道的鸡新城疫病毒F基因序列设计了一对引物,以鸡新城疫病毒HeB02分离株基因组为模板,通过RT-PCR扩增出了1·66kb左右的F基因片段,序列分析表明HeB02株F基因与国内标准强毒株F48E9及弱毒疫苗La Sota和Clone30的F基因核苷酸序列的同源性分别为88·1%、84·9%和83·8%。将HeB02株F基因插入真核表达载体pVAX1中,构建了真核表达质粒pSV-F,通过脂质体转染COS-7细胞,SDS-PAGE分析可见表达的特异蛋白条带;Western blot、ELISA和中和试验检测结果表明:真核表达的蛋白与抗新城疫病毒的抗体发生特异性反应,说明F蛋白具有很好的免疫原性。采用活体电击法以真核表达质粒pSV-F免疫3周龄SPF鸡,剂量为50μg/只,3周后加强免疫1次,5周后以100倍鸡胚感染剂量(EID)的F基因同源病毒对所有鸡进行攻毒,攻毒前后每周分别以喉拭子进行病毒分离和HI效价测定。结果显示对照组在攻毒前一直没有检测到抗体效价,攻毒后检测效价为3·0log2±1·40,并且于攻毒后第9天全部死亡;活疫苗组和实验组免疫后第2周检测到抗体效价,第5周最高,HI效价分别为8·3log2±1·30和7·2log2±1·23,攻毒1周后HI效价分别达9·8log2±1·55和8·9log2±1·77,极显著高于对照组(P<0·01)。免疫组未分离到新城疫病毒,对照组全部分离到新城疫病毒。表明所构建的F基因真核表达质粒可作为候选基因疫苗诱导鸡产生免疫保护反应。

Cloning of F Gene of Newcastle Disease Virus HeB02 Isolate and the Study of Its DNA Vaccine

In order to amplify F gene of NDV HeB02 strain, one pair of primers was designed according to the GenBank sequence, and a 1.66 kb F gene fragment was obtained by RT-PCR. Sequence analysis indicated that the homologies of the nucleotide sequence of HeB02 strain to those of F48 E9, La Sota and Clone30 strains were 88.1%, 84.9% and 83.8% respectively. The expression plasmid pSV-F was constructed by inserting the F gene into the pVAX1 vector, and transfected into the cultured COS 7 cell line via liposomes. The specific 5.9 kD protein was detected by SDS-PAGE and the immunogenicity of the expressed F protein was confirmed by Western blot, ELISA and neutralization test. 3 week-old SPF chickens were subcutaneously immunized twice at week 0 and 3 with 50 microg DNA of plasmid pSV-F by electroporration. 5 weeks later, all chickenss were challenged with 100 x EID50 of NDV HeB02 strain, 1 week post challenge all chickenss were sampled by larynx swabbing to isolate virus and the HI level of NDV was measured. The results indicated that the virus isolation was negtive in all vaccinated chickenss and positive in all control chickens. The HI titres reached to 8.3log2 +/- 1.30 and 7.2log2 +/- 1.23 induced by NDV vaccine and positive cells (pSV-F), respectivily, the HI titres induced by Control cells (pVAX1) was not detected. Furthermore, the HI titres reached to 9.8log2 +/- 1.55 and 8.9log2 +/- 1.77 in vaccinated group with NDV vaccine and positive cells (pSV-F), respectivily, were sinificantly higher than that of the control cells (pVAX1) immunized group( HI titers was 3.0 log2 +/- 1.40, P < 0.01) after challenge. These results showed that the plasmid pSV-F could be as a candidate of DNA vaccine to provide protective immune response against NDV infection.