猪瘟病毒E2基因的克隆及分子特性分析

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

为研究四川地区猪瘟病毒(classical swine fever virus,CSFV)及E_2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CSFVE_2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E_2基因的分子特性。结果显示,扩增的E_2基因大小为1 125bp,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 E_2的同源性较低,为87.6%。密码子偏向性分析显示,E_2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,E_2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,E_2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E_2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 E_2功能研究及后期基因工程苗的研制提供了基础数据。

To study the genetic variations of classical swine fever virus（CSFV）E_2 gene in Sichuan province,we used RT-PCR method to amplify E_2 gene and then cloned into pMD18-T vector,and strongly confirmed by PCR amplification,restriction digestion and sequencing,then the CSFV E_2 gene was analyzed by bioinformatics softwares.The results showed that the CSFV E_2 gene was 1 125 bp,and encoded a polypeptide of 375 amino acid residues.Moreover,the nucleobtide sequence of CSFV E_2 had higher homology with its homologous protein of other CSFV strains GXWZ02,CSFV/2.1,HEBZ,YC11 WB,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 HCLVE_2 gene（87.6%）.The ENc value of E_2 gene was 53.161,and there were distinctly differences of 33,25 and 25 codons frequency comparing CSFV E_2 with E.coli,yeast and human,respectively.Besides,there were total 34 rare codons in CSFVE_2 gene and serial rare codons being strung together.The results suggested that E_2 gene was not obvious in codonpreference,and was closest to eukaryotes in codon usage pattern.The nucleotides of E_2 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 E_2 function and the development of genetic engineering seedlings at later stages.