肺炎链球菌自溶素核酸疫苗的制备及其免疫原性研究

目的构建肺炎链球菌自溶素（pneumococcal autolysin，LytA）核酸疫苗，并研究其免疫原性。方法采用聚合酶链反应方法从肺炎链球菌基因组中克隆肺炎链球菌自溶素基因，然后将该基因插入到pVAX1真核表达载体中，构建表达LytA的重组质粒，制备成超螺旋比例大于90％的核酸疫苗。实验组：BALB／c小鼠5只，对其分3次肌内注射核酸疫苗（100μg）；对照组：BALB／c小鼠5只，肌内注射pVAX1空载体（100μg）3次。分别采集小鼠血清，采用间接酶联免疫吸附方法测定血清LytA特异性抗体水平。结果扩增出的LytA基因（957bp）与基因库中LytA的编码序列（957bp）一致。成功构建了LytA核酸疫苗。实验组小鼠的血清LytA抗体水平明显高于对照组，差异有统计学意义（P〈0．01）。结论成功克隆了LytA基因，制备了LytA核酸疫苗，后者可在小鼠体内诱导较强的特异性体液免疫反应。

Preparation of streptococcus pneumococcai autolysin DNA vaccine and study of its immunogenicity

Objective To construct pneumacoccal autolysin(LytA) DNA vaccine and to study its immunogenicity. Methods The LytA gene was amplified from the pneumococeal genome by polymerase chain reaction and then was inserted into the enkaryotic expression vector pVAX1 to construct the recombinant expression plasmid. The BALB/cmice were intramuscularly injected with LytA DNA vaccine(100 μg) in the experimental group(n = 5) or with pVAX1 (100 μg) in the control group(n =5) for 3 times respectively. The LytA-specific antibody level was measured by the enzyme-linked immunosorbent assay method. Results The DNA sequence of the cloned LytA (957 bp) was consistent with the GenBank data (957 bp) and the recombinant expression plasmid pVAX1-LytA was successfully constructed. The LytA-specific antibody level was significantly higher in the experimental group than in the control group (P < 0.01). Conclusions The LytA gene was successfully cloned. LytA DNA vaccine was successfully prepared which could induce powerful humoral immune reaction in mice. (Chin J Ophthalmol and Otorhinolaryngol,2009,9:212-214)