CAJ | 학술논문

目的探讨含有淋球菌PIB(Porin IB)与NspA(Neisseria surface protein A)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、108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,108TCID50/(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.
목적 탐토함유림구균PIB(Porin IB)여NspA(Neisseria surface protein A)기인적중조선병독재BALB/c소서체내적면역효과.방법 장30지자성BALB/c소서수궤분위5조,즉중조선병독pAdEasy-1-PN저、중、고제량조,pAdEasy-1대조조화공백대조조,재제0주화제4주이기내주사방식면역BALB/c소서량차,저、중、고조적면역제량분별위매지매차104、106、108조직세포반수감염량(TCID50).재면역개시적제0、3、5주수집혈청,병우제5주무균취비장,분리림파세포,채용매련면역흡부시험(ELISA)분별검측혈청중특이성PIB항체수평화특이성NspA항체수평,채용새서람(MTT)법검측특이성림파세포증식활성,채용파편응집시험화보체용균시험검측면역소서혈청적항균활성.응용t검험처리실험수거.결과 재중조선병독면역후적소서중균가검측출침대PIB항원화NspA항원적특이성세포면역반응화체액면역반응.재면역후적제3주화제5주,PIB항체수평(F치분별위285.72화564.83,균P＜ 0.01)화NspA항체수평(F치분별위521.57화542.61,균P＜ 0.01)재불동조별차이균유통계학의의.PIB화NspA특이성림파세포자격지수재불동조별중차이야존재통계학의의(F치분별위171.61화233.96,P＜ 0.01).면역효과여면역제량정정상관,108TCID50/(차·지)시교위합괄적면역제량.면역소서혈청가응집림구균,병가재보체삼여하장기살멸.결론 함유림구균PIB여NspA기인적중조선병독가재BALB/c소서체내산생특이성세포면역반응화체액면역반응,시잠재적림구균역묘.
Objective To evaluate the immunogenicity of a recombinant adenovirus containing the porin IB (PIB) gene and Neisseria 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-1-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 108 50％ tissue culture infective dose (TCID50) of the recombinant adenovirus pAdEasy-1-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 (MTT) 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-1-PN,and 108 TCID50 per dose proved to be the optimal dose for immunization.The sera from mice immunized with pAdEasy-1-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.