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目的 :原核细胞表达贝氏柯克斯体 34× 10 3 外膜蛋白基因 ,评价 34× 10 3 重组外膜蛋白的免疫保护性。方法 :用PCR从我国贝氏柯克斯体分离株的基因组中扩增 34× 10 3 外膜蛋白基因 ,用该基因与原核表达质粒重组 ,构建重组表达质粒 ;用IPTG诱导重组质粒在大肠杆菌细胞内表达重组蛋白。用重组蛋白皮下接种免疫BALB/c小鼠2次 ,在加强免疫后的第 2 8天 ,用ELISA法检测小鼠的血清特异性抗体水平以及作体外脾淋巴细胞增殖试验检查小鼠的特异性细胞免疫水平 ;用 10ID50 贝氏柯克斯体腹腔注射攻击免疫小鼠 ,攻击后第 7天病理学检查小鼠脏器的组织病变和染色镜检脾脏的贝氏柯克斯体。结果 :SDS PAGE和免疫印迹分析证明重组质粒转化的大肠杆菌产生了 34× 10 3 重组蛋白 ,重组蛋白能与贝氏柯克斯体感染小鼠血清发生特异性反应。ELISA法检测出免疫小鼠血清中有高滴度的抗重组蛋白抗体 ,免疫小鼠的体外淋巴细胞增殖水平显著高于非免疫小鼠。组织病理学检查发现免疫小鼠的肺、肝、脾无明显病变 ,仅个别小鼠脾脏存在少量柯克斯体 ,而非免疫小鼠的肺、肝、脾病变十分明显 ,脾脏肿大并含有大量的柯克斯体。结论 :重组 34× 10 3 外膜蛋白具有的免疫保护性 ,能够诱导机体产生有效的抗贝氏柯克斯体的体液和细胞免疫应答
OBJECTIVE: The prokaryotic expression of 34 × 10 3 Outer Membrane Proteins from Beauveria bassiana was performed to evaluate the immunoprotective properties of 34 × 10 3 recombinant Outer Membrane Proteins. Methods: 34 × 10 3 outer membrane protein genes were amplified by PCR from the genome of Beauveria bassiana isolates. The gene was recombined with prokaryotic expression plasmids to construct recombinant expression plasmids. IPTG was used to induce recombinant plasmids in Escherichia coli Intracellular expression of recombinant protein. BALB / c mice were immunized subcutaneously with the recombinant protein twice a day. On the 28th day after booster immunization, serum specific antibody levels of the mice were detected by ELISA and the specificity of the mouse was examined by in vitro proliferation of spleen lymphocytes Immunization of mice was induced by intraperitoneal injection of 10ID50 Beeswax coccus. Pathological examination was performed on the 7th day after the mice were sacrificed to examine the histopathology of the organs of mice and the microscopic examination of C. cynocephala in the spleen. Results: SDS PAGE and Western blot analysis showed that the recombinant plasmid transformed E. coli produced 34 × 10 3 recombinant protein, and the recombinant protein could specifically react with sera of mice infected with cercosporidium. Anti-recombinant protein antibody was detected by ELISA in sera of immunized mice. The proliferation of lymphocytes in immunized mice was significantly higher than that of non-immunized mice. Histopathological examination showed that there was no obvious lesion in the lung, liver and spleen of the immunized mice. Only a few Kirux bodies were found in the spleen of some mice, while the pathological changes of the lung, liver and spleen of the non-immunized mice were obvious. The spleen was enlarged and contained A lot of cx body. CONCLUSIONS: The recombinant 34x103 outer membrane protein possesses the immunoprotective ability to induce the body to produce an effective humoral and cellular immune response to C. bachmannii