人IL-12与结核分枝杆菌抗原ESAT-6联合基因疫苗的免疫效果观察

Immune effects research of interleukin 12 and Mycobacterium tuberculosis ESAT-6 antigen combined DNA immunization

摘要:

人白细胞介素12（IL-12）与结核分枝杆菌免疫优势抗原ESAT-6 真核表达质粒联合基因免疫，诱导免疫应答效果观察。近交系BALB/c小鼠，随机分组： A组（生理盐水对照）、B组（pcDNA3.1空质粒对照）、C组（BCG对照）、D组（pcESAT-6）和E组（pcIL-12＋pcESAT-6）。B、D、E质粒免疫组小鼠分别于胫前肌肌肉注射布比卡因（7.5g/L）和质粒的混和物（1∶4，100μL，含质粒70μg/次），A组小鼠肌肉注射生理盐水和布比卡因的混和物（1∶4，100μL），均间隔2周免疫一次，共免疫3次；末次免疫时，C组小鼠皮下注射BCG菌液，0.3mL/只，含106CFU/mL。末次免疫后14d和28d，各组小鼠分别取血分离血清用于总IgG测定，同时分离脾细胞，经TB-PPD刺激后检测脾细胞增殖（XTT比色法）活性和脾细胞培养上清液中γ干扰素（IFN-γ）、白介素4（IL-4）分泌水平。pcESAT-6质粒DNA单独免疫（D组）或与pcIL-12 质粒DNA联合免疫（E组），均能诱导小鼠产生特异性抗体，且抗体水平在末次加强免疫后14～28d 逐渐增加；但pcIL-12与pcESAT-6联合免疫后，特异性抗体水平较pcESAT-6单独免疫增加不明显 (P<0.05)。C、D、E组免疫小鼠脾细胞体外经TB-PPD刺激后，E组小鼠特异性淋巴细胞增殖活性和IFN-γ分泌水平明显强于C组和D组（P<0.05 ），而IL-4分泌水平相互间未发现明显差异。末次加强免疫后14～28d，E组小鼠脾细胞增殖活性维持在较高水平，而C组小鼠脾细胞增殖活性先低后高，D组则先高后低；IFN-γ诱生水平，E组最高，C组次之，D组最低。pcIL-12与pcESAT-6质粒DNA联合免疫后能刺激机体产生强烈的细胞免疫和稳定的体液免疫，在动物体内诱发的细胞免疫较ESAT-6或BCG单独免疫时均有明显增加并维持较长时间，此外联合免疫后诱导的体液免疫也较BCG免疫有明显增加。

Abstract:

To investigate the immune effects of combined DNA immunization of human interleukin 12 and Mycobacterium tuberculosis ESAT-6 antigen. Thirty BALB/c mice were divided into 5 groups. They were immunized inter-muscularly with 100μL solution of normal saline (group A), pcDNA3.1 control plasmid (group B), pcESAT-6 plasmid (group D) and pcIL-12+pcESAT-6 plasmid (group E), respectively, for three times with 2 weeks interval. At the last time immunization, group C mice were immunized intra-dermally with 106CFU BCG suspended in normal saline in a volume of 100μL. After the last immunization 14 and 28 days, groups of mice (three mice per group) were sacrificed and separated their serum and splenocytes at once, respectively. The sera were cryopreserved for antigen-specific antibody production tests with ELISA. The separated mice spleen cells were cultured with completed RPMI 1640 medium in vitro. Specific splenocytes proliferation responses to TB-PPD antigen were measured by XTT colorimetry after five days culture. Another part of the culture spleen cells were stimulated with TB-PPD antigen for three days, then collected the supernatants to determine IFN-γ and IL-4 secretions level by ELISA test as well, respectively. The results show that both ESAT-6 immunization and IL-12+ESAT-6 combined immunization induce significantly anti-TB-PPD antibody production. Further, the antigen-specific splenocytes proliferation and IFN-γ production level of IL-12+ESAT-6 combined immunization are higher than pcESAT-6 or BCG immunization alone (P<0.05). However, it displays no distinct differences of IL-4 secretion among ESAT-6, BCG and IL-12+ESAT-6 combined immunization groups. It indicates that ESAT-6 immunization combined with IL-12 could induce much stronger specific cellular immunity than only ESAT-6 or conventional BCG immunization. (Therefore, codelivery of an IL-12 scereting plasmid with M. tuberculosis antigen may be a potent strategy for enhancing cellular immunity against M. tuberculosis.