鲍曼不动杆菌三价抗原融合蛋白的制备及其免疫原性研究

Preparation of trivalent fusion protein from Acinetobacter baumannii and its immunogenicity study

目的制备鲍曼不动杆菌SmpA/Omp22/NlpA(1⁃128)三价抗原融合蛋白,免疫接种Balb/c小鼠后探究其免疫原性。方法通过人工合成结合PCR扩增法获取smpA/omp22/nlpA(1⁃384)融合基因,将该融合基因克隆到质粒载体pColdI中,构建重组质粒pColdI⁃SON。将该重组质粒转化E.coli BL21进行原核表达,并将纯化的重组蛋白与弗氏佐剂混合作为免疫原经背部皮下注射接种Balb/c小鼠,并于初次接种后第14、28天各加强免疫1次,同时设置佐剂对照组和PBS对照组。分别于末次免疫后第7、21天小鼠内眦取血分离血清,ELISA检测抗体滴度。分离免疫小鼠脾淋巴细胞,检测抗原刺激培养后脾细胞增殖活性及IFN⁃γ/IL⁃4的分泌情况。结果所构建的重组质粒大小约为5.9 kb,转化大肠杆菌后诱导表达并纯化出分子量约56 kDa的重组蛋白,免疫接种后在小鼠体内检测到高滴度的抗原特异性IgG抗体,小鼠脾淋巴细胞增殖活性明显高于佐剂及PBS对照组(P<0.001),免疫小鼠脾淋巴细胞经抗原刺激后所产生的IL⁃4水平明显高于对照组(P<0.01),而IFN⁃γ产生水平各组间无显著性差异。结论成功制备了鲍曼不动杆菌SmpA/Omp22/NlpA(1⁃128)融合蛋白,小鼠免疫接种后显示出较强免疫原性。

Objective To construct Acinetobacter baumannii SmpA/Omp22/NlpA(1⁃128)trivalent fusion protein and investigate its immunogenicity.Methods The smpA/omp22/nlpA(1⁃384)fusion gene was obtained by artificial synthesis and PCR amplification,the fusion gene fragment was cloned into plasmid pColdI and trans⁃formed into E.coli BL21 for prokaryotic expression.BALB/c mice were inoculated subcutaneously with the purified recombinant fusion protein formulated with Freund′s adjuvant three times.At the same time,the adjuvant control group and the PBS control group were set up.Blood was collected from the mice orbital canthus on day 7 and 21 after the last immunization,and the serum antibody titers were detected by ELISA.Spleen lymphocytes were isolated and stimulated by the recombinant fusion protein in vitro,the proliferation of lymphocytes and the release level of IFN⁃γand IL⁃4 were detected.Results A 5.9 kb recombinant plasmid was constructed and identified.The recom⁃binant plasmid can express a recombinant fusion protein with molecular weight about 56 kDa.Antigen specific anti⁃body IgG was detected in the serum of the recombinant fusion protein immunized mice.The proliferative activity of spleen lymphocyte from the recombinant fusion protein immunized mice was significantly higher than that from the adjuvant and PBS control group(P<0.001).The IL⁃4 release level of the spleen lymphocytes from the recombinant fusion protein immunized mice was significantly higher than that from the control group(P<0.01),while the IFN⁃γrelease level showed no significantly difference.Conclusion The SmpA/Omp22/NlpA(1⁃128)fusion protein of Aci⁃netobacter baumannii was successfully prepared and showed strong immunogenicity after mice immunization.