副猪嗜血杆菌转铁结合蛋白A基因的分段表达及其免疫原性分析

Expression and Immunogenicity of Transferrin-binding Protein A Gene of Haemophilus parasuis

本研究旨在获得副猪嗜血杆菌(Haemophilus parasuis,HPS)转铁结合蛋白A基因(tbpA)的表达产物(TbpA),并对其免疫原性进行分析鉴定。采用生物信息学方法预测HPS的TbpA抗原表位,根据GenBank上发表的HPS(SH0165株)tbpA的核苷酸序列设计合成3对引物,用PCR从安徽省HPS分离株(LJ3)tbpA中分段扩增出tbpA1、tbpA2和tbpA3,并连接到pET-32a(+)载体上,经BamHⅠ和EcoRⅠ双酶切鉴定和测序确认,将重组质粒转化大肠杆菌BL21(DE3)中,进行IPTG诱导表达。利用SDS-PAGE检测目的蛋白,Western blotting分析重组蛋白的免疫活性;通过小鼠免疫攻毒保护试验和血清杀菌力试验,鉴定重组蛋白的免疫原性以及诱导机体产生保护性免疫反应的能力。结果表明,从HPS(LJ3)tbpA中扩增出与预期设计的1 140、897、666bp大小相符的3个片段(tbpA1、tbpA2、tbpA3),扩增产物连接载体得到重组质粒,在大肠杆菌中实现表达,获得大小为62、54、44ku的目的蛋白(rTbpA1、rTbpA2、rTbpA3),均能与HPS阳性血清反应。rTbpA1与甲醛灭活菌体对小鼠免疫攻毒的保护率分别为20%和40%,但rTbpA1引起小鼠平均死亡时间显著延迟,兔抗rTbpA1与兔抗HPS(LJ3)血清具有同样显著的杀菌活性。结果显示,成功表达的rTbpA1、rTbpA2、rTbpA3均具有良好的反应原性,其中rTbpA1更具有良好的免疫原性以及诱导机体产生保护性免疫反应的能力,可望成为副猪嗜血杆菌病疫苗和血清学检测的候选成分。

An experiment was conducted to express transferrin-binding protein A（TbpA） of Haemophilus parasuis（HPS） and investigate its immunogenicity.Three pairs of specific primers were designed according to the tbpA gene sequence of HPS SH0165 strain from GenBank,three fragments of tbpA1,tbpA2 and tbpA3 containing multiple antigen epitopes were accquired by PCR and cloned into pET-32a（＋） vector respectively.It was confirmed that three fragments were inserted into the prokaryotic expression vector correctly by using Restriction endonuclease digestion and sequencing.The plasmids were transformed into E.coli BL21（DE3） and expressed by induction of IPTG.The specific proteins were determined by SDS-PAGE and their immune activity was analyzed by Western blotting.The immunogenicity of the proteins was analyzed by vaccination efficacy assessment in mice and bctericidal assays.The results showed that three fragments of 1 140,897 and 666 bp were amplified by PCR and expressed proteins with the sizes of 62,54 and 44 kD.The rTbpA1 can partly protect mice from the challenge of H.parasuis LJ3 strain and a significant bactericidal activity was also detected.These results indicated that the successfully expressed rTbpA1,rTbpA2 and rTbpA3 proteins were of effective reactogenicity,and the rTbpA1 could induces an immune response and might be useful as an antigen for vaccine and serological detection against Glsser＇s disease.