新城疫病毒融合蛋白裂解位点基因的分离克隆及在原核细胞中的表达

The Isolation, Cloning and Expression of the Fusion Protein Cleavage Site Gene of the Newcastle Disease Virus in the Protoryotic Cells

用ＳＰＦ鸡胚繁殖新城疫病毒（ＮＤＶ）Ｆ４６Ｅ９株（强毒）、ＬａＳｏｔａＥ４株（弱毒），对病毒进行纯化，抽提ＲＮＡ。用１对均为２７个碱基的引物进行ＲＴ－ＰＣＲ，扩增出了２个毒株的融合蛋白裂解位点（Ｆｃ）基因。将Ｆｃ基因定向克隆入ｐＵＣ１８的ＥｃｏＲＩ和ＳａｌⅠ位点之间，获得２个毒株Ｆｃ基因克隆ｐＵＣＦ４６Ｆｃ和ｐＵＣＬａＦｃ，用ＥｃｏＲＩ／ＳａｌⅠ双酶切法、ＰｓｔⅠ单酶切法、ＰＣＲ法和核酸探针法对其进行了鉴定。将鉴定好的ＬａＳｏｔａＥ４Ｆｃ基因定向导入表达载体质粒ｐＢＶ２２１，获得重组子ｐＢＶＬａＦｃ。ＰＣＲ和内切酶酶切法鉴定表明，Ｆｃ插入的位置和方向都正确无误。用Ｅ．ｃｏｌｉＤＨ５α通过热诱导法进行了表达。用ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ法检测了表达产物。结果表明，有１条能够与ＮＤＶ多抗反应的特异条带，分子量约１５７００，与预期大小一致。

The NDV F 46 E 9 and LaSotaE 4 strains were replicated in SPF chicken embyros, the virus in the allantoic fluid purified, and the virus RNA extracted. One pair of primers both 27b in length have been designed for RT PCR, which respectively correspond to the 45￣63 and 486￣504 nucleotide acids of the NDV Fc gene. The Fc genes of the two strains were amplified and cloned directionally into pUC18 between the Eco RI and Sal Ⅰ cleavage sites, getting the two Fc gene clones of pUCF 46 Fc and pUCLaFc. Using the methods of digestion with Eco RI/Sal Ⅰ and Pst Ⅰ, PCR, nucleic acid probe, the recombinants were identified, showing that the positions and oritentions were all correct. The Fc gene of the LaSotaE 4 was transfered into the expression vector pBV221 between the Eco RI and Sal Ⅰ sites, resulting in the recombinant pBVLaFc, and the clone was identified by restriction endonuclease analysis and PCR, proving that the foreign gene was in the correct position and oritention. The recombinant has been expressed in the E coli DH5α, and the expression product detected by SDS PAGE and Western blot methods. The results showed that a predicted 15 700 protein band, which could react with the NDV antiserum, suggesting that this peptide was the Fc gene expression product.