摘要
目的探讨含有淋球菌PIB（PorinIB）与NspA（Neisseria surface proteinA）基因的重组腺病毒在BALB／c小鼠体内的免疫效果。方法将30只雌性BALB／c小鼠随机分为5组，即重组腺病毒pAdEasy-1-PN低、中、高剂量组，pAdEasy-1对照组和空白对照组，在第0周和第4周以肌内注射方式免疫BALB／c小鼠两次，低、中、高组的免疫剂量分别为每只每次104、106、10。组织细胞半数感染量（TCID50）。在免疫开始的第0、3、5周收集血清，并于第5周无菌取脾脏，分离淋巴细胞，采用酶联免疫吸附试验（ELISA）分别检测血清中特异性PIB抗体水平和特异性NspA抗体水平，采用噻唑蓝（MTT）法检测特异性淋巴细胞增殖活性，采用玻片凝集试验和补体溶菌试验检测免疫小鼠血清的抗菌活性。应用t检验处理实验数据。结果在重组腺病毒免疫后的小鼠中均可检测出针对PIB抗原和NspA抗原的特异性细胞免疫反应和体液免疫反应。在免疫后的第3周和第5周，PIB抗体水平（F值分别为285．72和564．83，均P〈0．01）和NspA抗体水平（F值分别为521．57和542．61，均P〈0．01）在不同组别差异均有统计学意义。PIB和NspA特异性淋巴细胞刺激指数在不同组别中差异也存在统计学意义（F值分别为171．61和233．96，P〈0．01）。免疫效果与免疫剂量呈正相关，10^8TCID50／（次·只）是较为合适的免疫剂量。免疫小鼠血清可凝集淋球菌，并可在补体参与下将其杀灭。结论含有淋球菌PIB与NspA基因的重组腺病毒可在BALB／c小鼠体内产生特异性细胞免疫反应和体液免疫反应，是潜在的淋球菌疫苗。
Objective To evaluate the immunogenicity of a recombinant adenovirus containing the porin IB （PIB） gene and Neisseffa surface protein A （NspA） gene of N. gonorrhoeae in BALB/c mice. Methods A recombinant adenovirus containing the PIB gene and NspA gene of N. gonorrhoeae （pAdEasy-l-PN） was constructed in previous studies. Thirty BALB/c mice were randomly and equally divided into 5 groups： low-, medium- and high-dose experiment group intramuscularly immunized with 104, 106, and lOs 50% tissue culture infective dose （TCID50） of the recombinant adenovirus pAdEasy-l-PN, respectively, pAdEasy-1 control group immunized with 106 TCID50 of pAdEasy-1, blank control group immunized with sodium chloride physiological solution. Immunization was carried out twice at a 4-week interval. Serum samples were collected at 0, 3 and 5 weeks after the first immunization, and spleens were removed at 5 weeks followed by the isolation of spleen lymphocytes. Enzyme linked immunosorbent assay （ELISA） was performed to determine the serum levels of PIB- specific and NspA-specific antibodies, methyl thiazolyl tetrazolium （MTF） assay to evaluate the proliferaton activity of spleen lymphocytes after stimulation by the recombinant PIB and NspA antigens. A slide agglutination test was used to estimate the anti-bacterial activity of murine serum. The complement-dependent bacteriolytic activity of murine serum was also evaluated. Statistical analysis was carried out by t test. Results Both humoral and cellular immune response specific to PIB and NspA were elicited by the recombinant adenovirus in mice. At 3 and 5 weeks after the immunization, significant differences were observed in the serum levels of PIB-specific （F = 285.72, 564.83, respectively, both P 〈 0.01） and NspA-specific （F = 521.57, 542.61, respectively, both P 〈 0.01 ） antibodies. Also, the proliferation index was statistically different among these groups in spleen lymphocytes stimulated with PIB and NspA （F = 171.61, 233.96, respectively, both P 〈 0.01）. The vaccination efficiency was positively correlated with the inoculation dose of recombinant pAdEasy-l-PN, and l0s TCID50 per dose proved to be the optimal dose for immunization. The sera from mice immunized with pAdEasy-l-PN could agglutinate N. gonorrhoeae and kill it in the presence of complement. Conclusions The recombinant adenovirus pAdEasy-1- PN containing PIB and NspA genes could induce specific humoral and cellular immune response in mice, and may be a potential vaccine against N. gonorrhoeae.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2013年第5期328-331,共4页
Chinese Journal of Dermatology
