肺炎链球菌溶菌酶作为肺炎链球菌多价核酸疫苗候选抗原的保护效能评价

Evaluation of the protections of multivalent DNA vaccine of Streptococcus pneumoniae containing LytA gene

目的:肺炎链球菌溶菌酶(N-acetylmuramoyl-L-alanine amidase,LytA)作为肺炎链球菌多价核酸疫苗候选抗原的保护效能评价。方法:PCR技术从肺炎链球菌R6标准株扩增LytA基因序列,构建重组真核表达质粒pcDNA3.1-LytA,免疫印迹(western-blot)检测重组质粒在哺乳动物BHK-21细胞中的表达。与前期构建的肺炎链球菌表面蛋白A(Pneumococcal surface protein A,PspA)N端序列基因构建的质粒pcDNA3.1-PspA’核酸疫苗联合或单独肌肉注射免疫BABL/c小鼠,检测各组小鼠血清特异性抗体IgG水平,并观察免疫小鼠肺炎链球菌D39腹腔攻击21d生存情况。结果:ELISA检测发现3组(pcDNA3.1-LytA组,pcDNA3.1-PspA’组,pcDNA3.1-LytA+pcDNA3.1-PspA’组)实验组小鼠特异性IgG水平较空质粒对照组显著升高(P<0.001),pcDNA3.1-LytA+pcDNA3.1-PspA’组小鼠抗LytA特异性抗体IgG水平较pcDNA3.1-LytA组明显升高(P<0.01),但是与pcDNA3.1-PspA’组比较抗PspA特异性抗体IgG水平无显著差异(P>0.05)。小鼠攻击试验提示pcDNA3.1-LytA+pcDNA3.1-PspA’组小鼠中位生存时间较pcDNA3.1-LytA组及空质粒对照组显著延长(P<0.01),但与pcDNA3.1-PspA’组比较中位生存时间无显著差异(P>0.05),生存曲线相似。结论:LytA联合PspA’的肺炎链球菌多价核酸疫苗免疫效能并不优于单一抗原PspA’的肺炎链球菌核酸疫苗,LytA作为肺炎链球菌多价核酸疫苗的优势候选抗原组分之一有待进一步研究。

Objective：To evaluate the ability of the cooperative protection of the Strep to coccus pneumoniae multivalent DNA vaccine containing lytA gene（N-acetylmuramoyl-L-alanine amidase,LytA）and pspA＇gene（N fragment of pneumococcal surface protein A,PspA）against intraperitoneal Streptococcus pneumoniae D39 infection.Methods The genomic DNA of the standard Streptococcus pneumoniae strain D39 was isolated.LytA gene fragment was amplified by polymerase chain reaction（PCR）and cloned into the eukaryotic expression vector pcDNA3.1（＋）,which was transfected into BHK-21 cells using Lipofectamine TM2000,and then the expression of LytA protein was detected with Western-blot.Furthermore,three formats of recombinant plasmid DNA vaccines（pcDNA3.1-LytA＋pcDNA3.1-PspA＇,pcDNA3.1-PspA＇,pcDNA3.1-LytA）were used to inject intramuscularly BALB/c mice,with pcDNA3.1（＋）plasmid and PBS as controls.The levels of antibodies against PspA＇and LytA proteins were checked.Live times of intraperitoneal infection mice by Streptococcus pneumoniae D39 in BALB/c models were analysis.Results：The three DNA vaccines all producted high levels serum special IgG antibodies.The anti-LytA antibodies level elicited by the DNA vaccine pcDNA3.1-LytA ＋ pcDNA3.1-PspA＇was higher than the DNA vaccine pcDNA3.1-LytA（P〈0.01）,but the anti-PspA＇antibodies level elicited by the DNA vaccine pcDNA3.1-LytA ＋ pcDNA3.1-PspA＇had no significant statistics deviation（P〉0.05）.The result of the effect on intraperitoneal infection by Streptococcus pneumoniae D39 in BALB/c mice models showed the median live times in pcDNA3.1-LytA ＋ pcDNA3.1-PspA＇group was longer than pcDNA3.1-LytA group and the control groups（P〈0.01）,but no significant statistics deviation was found between pcDNA3.1-LytA ＋ pcDNA3.1-PspA＇group with pcDNA3.1-PspA＇group（P〉 0.05）.Conclusion：The multivalent DNA vaccine of Streptococcus pneumoniae containing LytA gene and pspA gene had no the higher performance protection than the DNA vaccine only containing pspA gene against Streptococcus pneumoniae.It is should be further evaluated that LytA gene is concluded as the one of advantages candidate antigens of the Streptococcus pneumoniae multivalent DNA vaccine.