猪瘟病毒石门株NS2-3基因片段的序列测定及比较

Sequencing and Comparison of NS2 3 Gene Fragment of Classical Swine Fever Virus Shimen Strain

根据猪瘟病毒Ｃ株的序列，以计算机辅助设计，化学合成１对引物（ＰＦ５６４８／ＰＲ６６０４），应用ＲＴＰＣＲ技术从感染猪血中成功地扩增了我国猪瘟病毒强毒石门株ＮＳ２３基因片段，大小为９５７ｂｐ，位于ＮＳ３基因的中部ＮＴＰａｓｅ和Ｈｅｌｉｃａｓｅ活性区。克隆后测序，结果表明该段基因产物具有解旋酶超家族全部七个特征性保守序列，包括共同的ＮＴＰ结合基序Ａ位点（ＧＸＧＫＴ／Ｓ）和Ｂ位点（３ｈｙ，２ｘ）Ｄ。序列同源性比较表明，石门株与日本的ＡＬＤ和ＧＰＥ－株同源性最高，与其它３株猪瘟病毒（Ｃ株、Ｂｒｅｓｃｉａ株和Ａｌｆｏｒｔ株）的同源性也很高，并与２株牛病毒性腹泻病毒（ＢＶＤＶ）（ＮＡＤＬ株和ＳＤ１株）也有较高的同源性，尤其是由核苷酸序列推导的氨基酸序列，同源性均大于９０％，是瘟病毒属基因组中最保守的区段。

A couple of CSFV specific primer (PF 5648 and PR 6604) was designed with the aid of computer primer designation software and synthesized based upon the relative conserved regions of published sequences of C strain. NS2 3 gene (p125 gene) fragment of CSFV Shimen strain was amplified successfully by RT PCR from the anticoagulant blood of infected pig. The product length is 957 bp, located in the central of NS3, a putative NTPase and helicase domain. The obtained PCR product was cloned and then sequenced. The sequence showed that this fragment contained all of seven typical conserved segments of helicase superfamily, including two common NTP binding motifs, namely, “A” site (GXGKT/S) and “B” site (3hy, 2x) D. Sequence homology analysis revealed that Shimen strain had the highest homology with Japanese strains (ALD and GPE -), and slightly lower homology with other three CSFV strains (C, BresciaandAlfort). Shimen strain had also significant homology with two BVDV strains (NADL and SD 1). The deduced amino acid sequence homology of Shimen strain with five CSFV and two BVDV strains was all upper than 90%. It is further confirmed that this fragment is the most conserved in pestivirus amino acid sequence. It is consistent with its essential function in replication and translation of virus genome and in processing of polyprotein precursor.