表达猪繁殖与呼吸综合征病毒GP5和M融合蛋白的重组腺病毒的构建及其对小鼠的免疫原性

Construction and immunogenicity of recombinant adenovirus co-expressing the GP5 and M protein of porcine reproduction and respriratory syndrome virus in mice

摘要:

利用口蹄疫病毒(FMDV)2A蛋白具有自我裂解的功能, 将其作为连接肽将猪繁殖与呼吸综合征病毒(PRRSV)的GP5和M蛋白编码基因串联, 置于复制缺陷型腺病毒载体的表达盒中, 通过一次转录和翻译, 可以同时实现2个蛋白的表达, 以发挥GP5蛋白的病毒中和优势和M蛋白的细胞免疫优势作用。分别利用RT-PCR、间接免疫荧光和Western blotting等方法, 对获得的重组腺病毒(rAd-GP5-2A-M)进行检测, 结果均证明GP5-2A-M蛋白不仅获得了正确表达, 而且能自我裂解为GP5和M蛋白。以单独表达GP5(rAd-GP5)、M(rAd-M)和融合表达GP5-M(rAd-GP5-M)的重组腺病毒为对照, 研究该重组腺病毒在小鼠体内诱导免疫应答情况, 结果表明, 虽然4种重组腺病毒均能诱导小鼠产生特异性抗体和细胞免疫反应, 但重组腺病毒rAd-GP5-2A-M所诱导产生的体液免疫和细胞免疫应答水平最高。本研究结果提示, 利用FMDV 2A蛋白酶的自动裂解功能构建PRRSV复合基因工程疫苗是一种切实可行的新策略, 也为构建其他动物病毒病的基因工程疫苗提供了新思路。

Abstract:

FMDV 2A peptide was introduced as a linker between GP5 and M protein of porcine reproduction and respiratory syndrome virus (PRRSV) to allow automatic self-cleavage the polyproteins. This strategy simultaneously displayed the neutralizing action of GP5 protein and cell-mediated immunity of M protein. We put them into the expression cassette of adenovirus vector. The results of RT-PCR, IFA and Western blotting showed that GP5 and M protein were not only expressed correctly, but also self-cleavaged and assemble heterodimers formation. To detect the advantages of rAd-GP5-2A-M, we also constructed some other recombinant adenoviruses (rAd-GP5, rAd-M and rAd-GP5-M) as control. After inoculated subcutaneously into BALB/c mice, the four recombinant adenoviruses can induce PRRSV-specific antibodies and cell-mediated immune response, but the level of humoral and cell-mediated immune response against PRRSV induced by rAd-GP5-2A-M is the strongest among the four recombinant adenoviruses. All of these suggested that it is possible to develop one multi-gene engineering vaccine utilizing FMDV 2A peptide, and also provided a novel strategy for developing other viral disease vaccine.