猪白细胞介素6基因的克隆及其作为核酸免疫佐剂的研究

Cloning of porcine interleukin-6 gene and its adjuvant effects on DNA vaccine

为研究猪白细胞介素6(pIL-6)作为核酸免疫佐剂的可行性,本研究采用RT-PCR方法,从猪外周血淋巴细胞中扩增出639 bp的pIL-6基因,编码212个氨基酸残基。将pIL-6基因克隆至真核表达载体pVAX构建重组质粒pVAX-IL6,并转染于COS-7细胞后,通过western blot检测到pIL-6在细胞中的表达。将含有猪传染性胃肠炎病毒(TGEV)S基因的重组真核表达质粒pVAX-S与pVAX-IL6混合后腿部肌肉注射6周龄NIH小鼠,通过间接ELISA以及流式细胞仪分别检测免疫小鼠血清中特异性TGEV IgG抗体和外周血T淋巴细胞亚群数量,结果表明pVAX-S+pVAX-IL6免疫组的特异性抗体水平与外周血T淋巴细胞亚群数量显著高于pVAX-S+pVAX对照免疫组(p<0.05),显示了pIL-6作为核酸免疫佐剂的良好应用前景。

To investigate the feasibility of using porcine interleukin-6 （plL-6） as adjuvant for DNA vaccination, the full-length cDNA of plL-6 was amplified from porcine peripheral blood lymphocyte by RT-PCR and ligated into pMD19-T vector. Sequence analysis showed that the cDNA of plL-6 was 639 bp in length and encoded 212 amino acids. The plL-6 gene was subcloned into pVAX1 to construct eukaryotic expression plasmid pVAX-IL6 which was transfected into COS-7 cells and the expression of plL6 was confirmed by western blot. Furthermore, Six-weeks-old NIH mouse were immunized intramuscularly with transmissible gastroenteritis virus （TGEV） recombinant plasmid pVAX-S＋pVAX-IL6 and pVAX-S＋pVAX, respectively, the anti-TGEV IgG antibody in routine serum and peripheral blood T lymphocyte subgroups were detected by indirect ELISA or flow cytometer. The results shown that the antibody titers and T cell proliferation in pVAX-S＋pVAX-IL6 immunized group were significantly increased （p〈0.05） than that of pVAX-S＋pVAX immunized group. Our results demonstrated that plL-6 is a promising adjuvant for improving immunogenicity of DNA vaccination.