论文部分内容阅读
目的纯化随机组合多表位恶性疟疾疫苗M.RCAg-1蛋白,并进行各项鉴定。方法对BL21(DE3)-M.RCAg-1/pDS-ex工程菌发酵产物进行纯化,对纯化的M.RCAg-1蛋白进行各种理化性质鉴定;采用Western blot法检测其反应原性;以不同剂量的M.RCAg-1蛋白免疫BALB/c小鼠及新西兰大白兔,采用间接ELISA法检测小鼠血清抗体效价;间接荧光免疫法(IFA)分析免疫血清对天然疟原虫抗原的识别;酶联免疫斑点试验检测特异性鼠T淋巴细胞的活化及细胞因子的分泌;体外生长抑制试验检测免疫兔血清对恶性疟原虫生长的影响。结果 M.RCAg-1蛋白表达量约为菌体总蛋白的30%,浓度为1.5 mg/ml,纯度可达95%左右,等电点为5.0;内毒素残留量均小于2.5 EU/mg,宿主蛋白残留量为0.08%,N-末端氨基酸序列正确;可与相应鼠单抗特异性结合;免疫小鼠血清抗体水平随着免疫次数和免疫剂量的增加而升高,20及30μg剂量组在末次免疫后,血清抗体滴度均可达1∶1 031 707;各免疫组小鼠血清均能识别恶性疟原虫天然抗原,且呈抗原剂量依赖性;10、20和30μg剂量组能刺激相应特异性脾淋巴细胞显著增殖(P<0.01)及分泌IFNγ和IL-4(P<0.01),并能较好地抑制疟原虫体外生长。结论纯化的M.RCAg-1蛋白理化性质稳定,具有较强的免疫原性,能激发可持续、高滴度的特异性抗体,并能诱发显著的T细胞应答,是极具潜力的候选疫苗蛋白,具有较好的开发前景。
Objective To purify the M.RCAg-1 protein of the pooled multi-epitope malaria vaccine and to identify it. Methods The fermented products of BL21 (DE3) -M.RCAg-1 / pDS-ex engineered bacteria were purified, and the purified M.RCAg-1 protein was identified by various physico-chemical properties. The reaction products were determined by Western blotting. BALB / c mice and New Zealand white rabbits were immunized with different doses of M.RCAg-1 protein and the serum antibody titers were detected by indirect ELISA. IFA was used to detect the antigen of the natural Plasmodium antigen. Enzyme-linked immunosorbent assay was used to detect the activation of specific murine T lymphocytes and the secretion of cytokines. In vitro growth inhibition assay was used to detect the effect of serum on the growth of Plasmodium falciparum. Results The expression level of M.RCAg-1 protein was about 30% of the total bacterial proteins, the concentration was 1.5 mg / ml, the purity was about 95% and the isoelectric point was 5.0. The endotoxin residues were less than 2.5 EU / mg, The host protein residue was 0.08%, the N-terminal amino acid sequence was correct, and it could specifically bind to the corresponding mouse monoclonal antibody. The level of serum antibody in immunized mice increased with the increase of immunization times and immunization doses. After the last immunization, the serum antibody titers reached 1: 1 031 707. The serum of all immunized mice could recognize the natural antigens of Plasmodium falciparum in antigen-dose-dependent manner. The 10, 20 and 30μg dose groups could stimulate the corresponding specific The spleen lymphocytes proliferated significantly (P <0.01) and secreted IFNγ and IL-4 (P <0.01), and inhibited the growth of Plasmodium in vitro. Conclusion The purified M.RCAg-1 protein is a promising candidate vaccine because of its stable physicochemical properties, strong immunogenicity, ability to stimulate specific, sustained, high-titer antibodies and to induce significant T cell responses Protein, has a good development prospects.