猪瘟病毒E2基因的克隆及分子特性分析

为研究四川地区猪瘟病毒（classical swine fever virus，CSFV）及E2基因的遗传变异情况，试验对CSFV阳性病料抽提RNA，通过RT-PCR扩增CSFV E2基因并将其克隆到pMD18-T载体，经菌落PCR、双酶切、序列测定后利用生物信息学软件分析了E2基因的分子特性。结果显示，扩增的E2基因大小为1 125 bp，编码375个氨基酸，与GenBank中CSFV GXWZ02、CSFV/2.1、HEBZ、YC11WB、SXYL2006、0406/CH/01/TWN亲缘关系较近，核苷酸同源性分别为98.3%、96.5%、94.7%、92.4%、92.1%和90.9%，而与疫苗株HCLV E2的同源性较低，为87.6%。密码子偏向性分析显示，E2基因的ENc值为53.161，密码子使用频率与大肠杆菌、酵母菌和人差异较大的分别有33、25和25个；此外，E2基因共有34个稀有密码子，且有多个稀有密码子多次串联成簇的情况出现。结果表明，E2基因在密码子使用中整体上不存在较明显的偏向性，且密码子使用模式更接近真核生物，本试验克隆的E2基因与疫苗株相比，部分核苷酸出现了变异，但主要抗原域氨基酸未发生改变，该结果为进一步开展CSFV E2功能研究及后期基因工程苗的研制提供了基础数据。

Cloning and Molecular Characterization Analysis of E2 Gene of Classical Swine Fever Virus

To study the genetic variations of classical swine fever virus (CSFV) E2 gene in Sichuan province,we used RT-PCR method to amplify E2 gene and then cloned into pMD18-T vector,and strongly confirmed by PCR amplification,restriction digestion and sequencing,then the CSFV E2 gene was analyzed by bioinformatics softwares.The results showed that the CSFV E2 gene was 1 125 bp,and encoded a polypeptide of 375 amino acid residues.Moreover,the nucleobtide sequence of CSFV E2 had higher homology with its homologous protein of other CSFV strains GXWZ02,CSFV/2.1,HEBZ,YC11WB,SXYL2006 and 0406/CH/01/TWN in GenBank,the homologies were 98.3%,96.5%,94.7%,92.4%,92.1% and 90.9%,respectively,but it had lower homology with HCLV E2 gene (87.6%).The ENc value of E2 gene was 53.161,and there were distinctly differences of 33,25 and 25 codons frequency comparing CSFV E2 with E.coli,yeast and human,respectively.Besides,there were total 34 rare codons in CSFV E2 gene and serial rare codons being strung together.The results suggested that E2 gene was not obvious in codon preference,and was closest to eukaryotes in codon usage pattern.The nucleotides of E2 gene was partially mutated compared to vaccine strains,but the main antigenic domain didn't change.The results provided basic data for the further study of CSFV E2 function and the development of genetic engineering seedlings at later stages.