CPSIT_p7重组表达产物免疫原性检测及其在鹦鹉热嗜衣原体持续性感染中表达的研究

Immunogenicity analysis of a recombinant protein CPSIT_p7 and its expression in HeLa cells during persistent Chlamydophila psittaci infection

目的：构建鹦鹉热嗜衣原体（ Chlamydophila psittaci，Cps） CPSIT_p7原核表达载体，表达并纯化CPSIT_p7，检测其免疫原性，观察其在持续性感染过程中的mRNA和蛋白动态表达情况。方法原核表达和纯化His-CPSIT_p7融合蛋白，免疫BALB/c小鼠制备多克隆抗体，ELISA分析His-CPSIT_p7蛋白的免疫原性。采用青霉素与Cps共同处理细胞来制备持续性感染模型， RT-PCR和Western blot检测CPSIT_p7在其持续性感染过程中的表达情况。结果成功构建了pET30a-CPSIT_p7原核表达载体，并表达和纯化较稳定的重组蛋白；该蛋白免疫小鼠40 d后，ELISA 检测血清特异性抗体效价为1∶1000000；RT-PCR和Western blot 结果显示在Cps急性感染与持续性感染过程中CPSIT_p7 mRNA和蛋白表达水平均呈时间依赖性增加，感染后mRNA在36 h表达量达到高峰，之后急性感染呈时间依赖性下降，而持续性感染CPSIT_p7 mRNA高表达水平至少持续到60 h。结论成功克隆表达了His-CPSIT_p7，该蛋白具有较好的免疫原性；CPSIT_p7基因和蛋白在Cps持续性感染中呈高表达。

Objective To construct a prokaryotic expression plasmid for CPSIT_p7 gene from Chlamydophila psittaci （ Cps） 6BC strain and to evaluate immunogenicity of the recombinant protein His-CPSIT_p7 and detect its dynamic expression at mRNA and protein levels in HeLa cells during persistent Cps infection.Methods The fusion protein His-CPSIT_p7 was expressed in E.coli BL21 and purified by Ni-NTA affinity chromatography .BALB/c mice were immunized with the recombinant protein to prepare polyclonal antibody for evaluation of the immunogenicity of His-CPSIT_p7 by ELISA.Penicillin sodium was used to establish a model of Cps persistence infection .RT-PCR and Western blot assay were performed to de-tect the expression of CPSIT_p7 at mRNA and protein levels during Cps persistent infection .Results The fusion protein His-CPSIT_p7 was successfully expressed with the use of constructed recombinant expression plasmid pET30 a-CPSIT_p7 and purified .ELISA result showed that the specific antibody titer against CPSIT_p7 reached 1 ∶1 000 000 on the 40th days after immunization .The expression of CPSIT_p7 at mRNA and protein levels were increased in a time-dependent manner in Cps-infected HeLa cells .The peak of mRNA level was reached at the time point of 36 hours after infection , followed by a time-dependent decrease during Cps acute infection .However , the expression of CPSIT_p7 at mRNA and protein levels were not decreased until 60 hours after infection during Cps persistent infection .Conclusion His-CPSIT_p7 protein was suc-cessfully expressed in the prokaryotic expression system and purified , showing an advantage of good immuno-genicity.Highly expressed CPSIT_p7 at mRNA and protein levels were detected during Cps persistent infection.