随机组合多表位恶性疟疾疫苗M.RCAg-1蛋白的纯化及鉴定

Purification and Identification of Randomly Recombinant Multiepitope Protein M.RCAg-1 of Plasmodium falciparum Vaccine

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 and identify the randomly recombinant multiepitope protein M.RCAg-1 of Plasmodium falciparum vaccine.Methods The fermentation product of recombinant E.coli BL21（DE3）-M.RCAg-1 / pDS-ex was purified,then investigated for physic-chemical property and determined for reactogenicity by Western blot.BALB / c mice and New Zealand rabbits were immunized with M.RCAg-1 protein at various dosages,and determined for antibody titer in sera by indirect ELISA.The recognition of natural parasite antigen with the immune sera was determined by IFA.The activation of mouse specific T lymphocytes and secretion of cytokines were determined by enzyme-linked immunosorbent spot test.The effect on growth of Plasmodium falciparum was observed by growth inhibition test in vitro.Results The M.RCAg-1 protein,with an isoelectric point of 5.0,contained about 30% of total somatic protein,reached a purity of more than 95% and showed specific reaction with mouse specific monoclonal antibody,of which the concentration was 1.5 mg / ml,the residual endotoxin content was less than 2.5 EU / mg,the residual host protein content of 0.08%,and the amino acid sequence at N-terminus was correct.The serum antibody titer increased with the increasing doses and dosages of M.RCAg-1 protein,while those after last immunization with 20 and 30 μg of M.RCAg-1 protein reached 1 ∶ 1 031 707.All the immune sera of mice recognized the natural antigen of Plasmodium falciparum in a dose-dependent mode,while those induced by 10,20 and 30 μg of M.RCAg-1 protein stimulated the proliferation of specific splenic lymphocytes（P 0.01） as well as secretions of IFNγ and IL-4（both P 0.01）,and inhibited the growth of Plasmodium falciparum in vitro significantly.Conclusion The purified M.RCAg-1 protein showed stable physic-chemical property and high immunogenicity,stimulated persistent high titer specific antibody and induced significant T cell response,which might be used as a potential candidate vaccine of good prospect of development.