牛疱疹病毒Ⅰ型gD蛋白的表达、纯化及抗原性检测

Expression,purification and antigenicity detection of BoHV-1 gD protein

为了能表达出具有良好免疫原性的牛疱疹病毒Ⅰ型(BoHV-1)gD蛋白,为进一步建立BoHV-1检测方法奠定基础,试验将BoHV-1 gD蛋白的基因片段通过PCR扩增、连接、转化的方法连接到pGEX-6P-1载体上,经过单双酶切鉴定、PCR鉴定后测序。测序结果与NCBI上公布的BoHV-1 gD蛋白的氨基酸序列进行对比,并通过SDS-PAGE、Western-blot以及ELISA检测方法对蛋白质表达情况、抗原性进行检测分析。结果表明:gD基因成功插入pMD18-T载体中,将测序结果与已经发表在NCBI上的BoHV-1 gD蛋白基因比对,相似性为98%,共有19个碱基发生突变,并无碱基插入或缺失。通过软件翻译与原始氨基酸序列(AFB76672.1)进行比对,共有8个氨基酸发生突变。将gD基因插入pGEX-6p-1载体中,命名为pGEX-6p-gD质粒,构建的pGEX-6P-gD/BL21(DE3)重组菌表达出的蛋白质与目的蛋白质大小一致,且该蛋白质免疫后小鼠血清特异性识别BoHV-1。说明本试验成功表达出具有部分免疫原性的BoHV-1重组gD蛋白。

In order to express good immunogenicity Bovine herpesvirus type 1(BoHV-1)gD protein,and further establish BoHV-1 detection method to lay a foundation,the gene fragment of BoHV-1 gD protein was connected to pGEX-6P-1 vector by PCR amplification,binding and transformation,and sequenced after single and double enzyme digestion identification and PCR identification.The sequencing results were compared with the amino acid sequence of BoHV-1 gD protein published on NCBI,and the antigenicity of protein expression was detected and analyzed by SDS-PAGE,western-blot and ELISA.The results showed that gD gene was successfully inserted into pMD18-T vector,and the sequencing results were 98%similar to those of BoHV-1 gD gene published on NCBI.A total of 19 bases were mutated without base insertion or deletion.Compared with the original amino acid sequence(AFB76672.1)through software translation,a total of 8 amino acids were mutated.gD gene was inserted into pGEX-6P-1 vector and named pGEx-6P-gD plasmid.The protein expressed by recombinant pGEX-6P-gD/BL21(DE3)constructed was consistent in size with the target protein,and the protein was specifically identified as BoHV-1 in serum of mice after immunization.The results indicated that BoHV-1 recombinant gD protein with partial immunogenicity was successfully expressed.