CVB3-VP1基因免疫诱导特异性抗病毒免疫应答及保护作用

目的 :构建表达柯萨奇病毒B3(CVB3)主要包膜蛋白VP1的基因疫苗 ,并研究该疫苗诱导CVB3特异性免疫应答及免疫保护的作用。方法 :抽提CVB3RNA ,以RT PCR扩增VP1基因 ,克隆于真核表达载体 pcDNA3中 ,构建质粒pcDNA3 VP1。将该质粒转染Hela细胞 ,观察其表达情况 ;以 5 0 μgpcDNA3 VP1质粒DNA肌注免疫BALB/c小鼠 3次 ,检测CVB3特异性体液和细胞免疫应答。间隔 4wk以 5×LD50 的CVB3攻击小鼠 ,观察攻击后小鼠的存活情况。结果 :构建了重组质粒 pcDNA3 VP1,并在体外获得有效表达。以该质粒肌肉免疫BALB/c小鼠 ,可诱生高水平的IgM和IgG ,VP1多肽特异性淋巴细胞增殖反应及CTL活性均显著高于 pcDNA3免疫的对照组。病毒攻击试验表明 ,pcDNA3 VP1免疫组33.3%小鼠可长期存活 ,其心肌组织未见明显的病理学改变 ;而对照小鼠平均仅存活 6 .7d ,心肌显示大量的局灶性坏死和炎性细胞浸润。结论 :pcDNA3 VP1免疫可诱生CVB3特异性体液及细胞免疫应答 ,保护免疫小鼠抵抗CVB3的致死性攻击

Protective immunoresponse to CVB3 induced by gene immunization with pcDNA3-VP1

AIM: To induce Coxsackie virus B type 3 (CVB3)-specific immune response by using a DNA vaccine containing CVB3-VP1 and to observe its protection against CVB3 challenge. METHODS: The gene coding for VP1 was obtained by RT-PCR and then was cloned into plasmid pcDNA3 to construct pcDNA3-VP1. In-vitro expression of VP1 was performed by transfection of pcDNA3-VP1 into Hela cells. Expressed product was detected by ELISA. BALB/c mice were immunized intramuscularly with 50 microg DNA three times, and challenged by 5xLD(50) CVB3 four weeks after the last immunization. RESULTS: pcDNA3-VP1 had been constructed and the expression product was detected in the culture supernatant of Hela cells 24 hours after transfection. CVB3-specific IgM and IgG elicited in the mice immunized with pcDNA3-VP1 were significantly higher than those in the control mice immunized with pcDNA3. Specific proliferation of the splenic lymphocytes and activity of CVB3-specific CTLs from the pcDNA3-VP1 immunized mice were much stronger than those in the controls. pcDNA3-VP1 could protect 33.3% mice from lethal CVB3 challenge, while control mice only survived 6.7 days. Infiltration of inflammatory cells or unusual proliferation of connective tissue, indicating ongoing myocarditis or fibrosis, were not found in pcDNA3-VP1 immunized mice, but did exist in control mice. CONCLUSION: Intramuscular immunization with pcDNA3-VP1 may be a promising approach against CVB3 infection.