猪链球菌2型表面蛋白sp融合基因的构建与高效表达

Construction and expression of sp fusion gene of Streptococcus suis serotype 2

利用PCR方法从猪链球菌2型四川资阳分离株449-1扩增出sp基因,克隆到pMD18-T载体中,构建出克隆载体pMD18T-sp。以SalⅠ和BamHⅠ从pMD18T-sp中切下目的sp基因片段并克隆到质粒pMAL-p2X中,构建重组表达质粒pMALp2X-sp,转化宿主菌TB1中进行诱导表达。分析表明sp基因序列比GenBank报道序列AY864331少了270 bp;SDS-PAGE电泳检测结果表明,重组菌株表达出了136 Ku左右的目的蛋白MBP-Sao,目的蛋白为可溶表达,约占菌体蛋白总量的12.3%。表达的蛋白可用Amylose树脂纯化。将纯化的融合蛋白MBP-Sao免疫家兔,所得抗血清经ELISA检测,结果显示融合蛋白MBP-Sao包板抗体效价达1:16 000,且与猪链球菌2型和9型呈阳性反应,而与沙门氏杆菌及巴氏杆菌呈阴性反应。本试验对猪链球菌2型表面蛋白Sao的高效表达及其免疫原性进行了初步研究,为进一步研究猪链球菌2型表面蛋白Sao的结构和功能奠定了基础。

Streptococcus suis serotype 2 is an important zoonotic pathogen. The Sao protein encoded by sp gene is a newly identified surface protein of S.suis type 2. The sp gene was amplified by PCR from S.su/s type 2 449-1 isolated from Sichuan province, and cloned into pMD18-T. Sequencing of the cloned gene revealed that the sp gene of strain 449-1 was 270 bp shorter than that of the foreign strain S735 （GenBank accession AY864331）. The sp gene was released from the plasmid pMD 18-T-sp by Sa/I and BamH I, and subcloned into expression vector pMAL-p2x. The recombinant plasmid was transformed into E.coli TB1, then induced by IPTG at 35 ~C. The fusion protein MBP-Sao had a molecular weight approximately 136 Ku, which accounted for 12.3 % of the total bacteria protein. The protein was purified using an amylose column and used to immunize the rabbits. High antibody yield with a titre of 1：16 000 was detected by ELISA. The antisera collected from the immunized rabbits reacted with Streptococcus suis serotype 2 and 9, but not with Salmonella and Pasteurella. This study provides foundation for further research on the structure and fimction of the protein Sao of S.suis type 2.