中文摘要：

为了探索牛乳腺炎无乳链球菌磷酸甘油酸激酶（Phosphoglycerate kinase,Pgk）基因编码蛋白的抗原性,根据GenBank公布的牛源无乳链球菌Pgk基因序列,设计并合成一对引物,通过PCR扩增出Pgk蛋白抗原优势区的编码序列,并对其进行克隆、测序、转化、诱导表达及抗原性鉴定。结果显示克隆的Pgk基因序列含有984 bp,编码328个氨基酸残基。临床分离株与GenBank上公布的B群无乳链球菌菌株Pgk基因（AE009948）核苷酸序列同源性为99.09%,氨基酸序列同源性为99.69%。将扩增的Pgk基因片段克隆至原核表达载体pET30a（＋）中,构建重组表达载体Pgk-pET30a（＋）,再将筛选的阳性重组质粒转化至BL21工程菌,经IPTG诱导获得部分可溶性表达Pgk重组蛋白。经Ni2＋亲和层析柱纯化得到纯度在90%以上的蛋白,经Western blot分析结果表明,重组蛋白具有良好的抗原性,为进一步探索其对奶牛的免疫原性研究奠定了良好的实验基础。

英文摘要：

A pair of primers were designed and synthesized according to Pgk gene sequence of bovine mastitis streptococcus agalactiae published on Genbank.Antigenic dominant region of Pgk gene was amplified by PCR.The sequencing of PCR product showed that the cloned region of Pgk gene is 984bp in length,which encoding 328 amino acid residues.The nucleotide and amino acid identities of the partial Pgk gene between the clinical strain and Streptococcus agalactiae group B strain（AE009948） are 99.09% and 99.69%,respectively.The PCR product was cloned into prokaryotic expression vector pET30a（＋） and generated recombinant expression vector pET30a（＋）-Pgk,recombinant plasmid was then transformed to E.coli BL21 and induced by IPTG.The expressed partial Pgk protein was purified by Ni2＋ affinity column.The result of Western blot analysis showed that the recombinant protein could specifically react with antibodies against bovine mastitis Streptococcus agalactiae and may be used to analyse in further for immunogenicity.