肺炎链球菌疫苗候选蛋白质谷氨酰胺tRNA合成酶的保守性与抗原性分析  

Analysis of conservation and antigenicity of candidate protein in vaccine for Streptococcus Pneumoniae: Glutamyl tRNA synthetase

作　　者：闵迅[1] 黄美容[1] 黄健[1] 董杰[1] 陈特[1] 王虹[1] 钟文[1] 尹一兵[1] 

机构地区：[1]重庆医科大学检验系临床检验诊断学实验室,重庆400016

出　　处：《临床检验杂志》2012年第6期442-446,共5页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金项目(30970110)

摘　　要：目的分析作为肺炎链球菌(Streptococcus pneumoniae,S.pn)疫苗候选靶点的谷氨酰胺tRNA合成酶(glutamyl tRNA syn-thetase,Gts)的保守性和抗原性,并评价Gts抗原诱导产生的抗体在体内是否存在年龄依赖性。方法 PCR扩增S.pn D39标准菌株的Gts基因,构建pET32a(+)-Gts原核表达质粒并测序。扩增S.pn不同血清型的Gts基因,测序后比对分析其保守性。表达Gts重组蛋白质,并进行纯化和western blot鉴定。用Gts重组蛋白质免疫BALB/c小鼠制备抗Gts的多克隆抗体并测定其效价。western blot分析Gts在不同血清型S.pn中的表达情况。ELISA法测定本地区不同年龄段的健康人及患者血清中抗Gts抗体的效价。结果构建了以D39血清型S.pn为DNA模版的pET32a(+)-Gts表达质粒,其测序结果与预期相符。8株不同血清型S.pn的Gts基因编码区大小与D39菌株一致,约为1 461 bp;经测序后序列比对,其基因一致性>99.0%。经Ni柱纯化得到纯度>95%的Gts重组蛋白质,且能与抗His tag标签抗体特异性结合。采用Gts蛋白质经腹腔免疫小鼠后,其血清抗Gts抗体滴度高达1.024×106(5.12×105,4.096×106),与对照组比较有显著差异(P<0.01)。western blot分析证实,8种不同血清型S.pn全菌裂解物均在Mr约55.9×103处出现特异性反应条带。在不同年龄段的健康人群及患者血清中,均产生高滴度的抗Gts抗体,且随着年龄增加而递增。结论 Gts重组蛋白质免疫原性好,在不同血清型的S.pn中均高度保守,且诱导产生的抗体反应存在年龄依赖性,是一个良好的候选疫苗靶点。Objective To investigate the conservation and antigenicity of glutamyl tRNA synthetase （ Gts）, the candidate target site of potential vaccine for Streptococcus pneumoniae（ S. pn） , and estimate the age-dependence of the antibody to Gts in human body. Meth- ods Gts gene from different S. pn serotypes was amplified with PCR, and its conservation was analyzed by sequence alignment. Re- combinant Gts protein was obtained through prokaryotic expression system, and was further purified and identified with western blot- ting. BALB/c mice were immunized with the recombinant protein to prepare the polyclonal antibody to Gts protein and the titer of the antibody was determined. The expression levels of Gts protein in S. pn with different serotypes were determined by western blot. The ti- ters of the antibody to Gts in the serum from the healthy subjects with different age and the patients with S. pn infection were determined by ELISA. Results The integrated plasmid pET32a（ ＋ ） -Gts was constructed successfully with S. pn serotype D39 DNA as template, and the recombinant protein Gts with high purity （ more than 95% ） was obtained after purification of Ni affinity chromatography. The titer of the antibody to Gts in the immunized mice serum reached to 1. 024 x 106 （5.12 x 105 ,4. 096 x 106 ）. The gene coding region with identical 1 461 bp of length presented in all the 8 different S. pn serotypes, and the sequence homology was 99%. western blotting showed the same band with Mr 55 900 presented in the lysate of all the 8 serotypes of S. pn. High titer of antibodies to Gts was detecta- ble in the sera from healthy subjects and S. pn-infected patients, and the titer increased with age. Conclusion The recombinant pro- tein Gts with high immunogenicity and conservation induced the antibody to Gts with high titer in different serotypes of S. pn, indicating that Gts may be a promising candidate protein for vaccine of S. pn.

关 键 词：肺炎链球菌 谷氨酰胺tRNA合成酶 保守性 抗原性 疫苗 

分 类 号：R392.7[医药卫生—免疫学]