法氏囊炎病毒VP2基因高变区片段的克隆与鉴定

[目的]研究法氏囊病毒VP2蛋白的抗原表位在机体免疫应答中的作用和特点。[方法]以接种传染性法氏囊病毒(IBDV)的鸡胚尿囊液为模板,根据Genbank中登录的IBDV的VP2蛋白基因的全序列设计引物,通过RT-PCR扩增获得VP2高变区部分片段,将其克隆到原核表达质粒PGEX-4T-1中,经酶切鉴定后,转化大肠杆菌Rosetta(DE3)BL21,筛选阳性克隆并对其进行测序鉴定。[结果]通过PCR扩增获得一个504bp的扩增片段,序列分析结果表明它与GenBank中登录的IBDV的VP2蛋白基因的核苷酸序列同源性达99.8%,其氨基酸序列的同源性为99.4%,说明各毒株间高变区基因序列保守性很高。[结论]法氏囊炎病毒毒株在两个大小亲水区内的氨基酸都非常保守。

Cloning and Identification of a Fragment of Hypervariable region from IBEV-VP2 gene

[Objective] The research aimed to study the functions and characters of the antigen epitope of IBDV-VP2 protein in immune response. [Method] The cDNA fragment of IBDV-VP2 gene was amplified from the allantoic fluid of chick embryo mRNA by RT-PCR using the primers designed from full length sequence of IBDV-VP2 gene reported in GenBank. The fragment was inserted into pGEX-4T-1 expressive vector,then transformed into Escherichia coli BL-21 after identified with enzyme restriction,and then sequenced through screening the positive clones. [Result] A cDNA fragment of 480 bp was amplified by RT-PCR. Sequence analysis showed that it shared 99.8% homology with the nucleotide sequence of IBDV-VP2 gene reported in GenBank and 99.4% homology of amino acid sequence,which indicated that the gene sequence of hypervariable region from every strain was highly conservative. [Conclusion] The amino acid sequence of IBDV strain was conservative in the two hydrophilic regions.