口蹄疫病毒VP1与3ABC基因片段的克隆及表达

Cloning and Expression of VP1 and 3ABC Genes of Foot-and-Mouth Disease Virus Type O

根据GenBank中注册的O型口蹄疫病毒(FMDV)VP1和3ABC基因序列,设计了2对引物.采用RT-PCR方法从FMDV分离株O/HK/2001扩增得到VP1和3ABC基因.经对它们的核苷酸序列测序和同源性比较,显示与14株O型FMDV参考毒株中的O/HKN/2002同源性最高(分别为98.3%和98.6%),并且在VP1决定各种亚型FM-DV免疫原性的主要抗原决定族的144、148、154和208位关键氨基酸,比对均未发现变异.通过酶切将VP1和3ABC基因分别亚克隆到4个表达载体pET-30a、pPROEX HTb、pQE30和pQE9中构成重组表达质粒,利用IPTG诱导表达,经SDS电泳分析表明这些重组质粒在相应表达宿主菌BL21、DH5α和M15中,除pQE30-3ABC和pQE9-3ABC外均获得了表达,其中pET-30a和pPROEX HTb载体表达量较高.

pairs of primer were designed based on VP1 and 3ABC genes of foot-and-mouth disease vims（FMDV） serotype O which were registered in the GenBankTM. VP1 and 3ABC genes were amplified from the O/HK/2001 strain of FMDV by RT-PCR. Nueleotide analysis and homology comparison indicated that the sequences shared the highest homology with that of the strain O/HKN/2002 among the 14 strains. No variation was found on the antigenic sites of 144,148,154 and 208 of the key amino acids in the VP1 gene which determines various subtypes of FMDV. Then the VP1 and 3ABC genes were subcloned into four expression vectors pET-30a, pPROEX HTb, pQE30 and pQE30 to construct expression plasmids. IPTG induced expression and SDS electrophoresis indicated that except pQE30-3ABC and pQE9-3ABC,the others can be expressed in the bacteria of BL21, DH5α and M15, pET-30a and pPRO-EX HTb vector＇ s expression are the highest among them.