CAJ | 학술논문

目的研究载体融合肽段对人工重组多表位蛋白M.RCAg-1构象、免疫原性及体外保护性的影响.方法将人工多表位抗原M.RCAg-1基因通过不同的酶切位点克隆到原核表达载体pDS-ex上,从而获得带有不同载体融合标签或没有标签的蛋白,表达产物经纯化得到P312-1、P312-2、P312-3这3种多表位蛋白,利用生物信息学和色谱技术对制备的3种蛋白的二级及三级结构进行了分析,并将这3种纯度大于95％的重组蛋白与弗氏佐剂乳化后分别免疫接种小鼠和新西兰白兔,免疫后血清按常规进行ELISA间接法检测抗体滴度,采用酶联免疫斑点试验(ELISPOT)检测分泌细胞因子的特异性淋巴细胞克隆数,同时进行了间接免疫荧光分析(IFA)以及对体外培养的恶性疟原虫的生长抑制试验(GLA).结果 P312-1、P312-2、P312-3这3种多表位蛋白在动物模型体内诱导的抗体滴度水平并无差异(P＞0.05),但蛋白免疫小鼠后却诱导了不同类型的T细胞反应,而且3种蛋白免疫血清IgG体外生长抑制率出现明显差异(分别为93.9％、14.7％、54.3％).利用生物信息学和色谱技术分析,发现从不同表达载体制备的重组蛋白在二级和三级结构上出现极大差异,预测蛋白质分子的空间构象发生了改变,而且一些表位在分子中的位置也有明显的改变.结论不同的载体肽融合表达对多表位蛋白M.RCAg-1结构及免疫活性的影响会产生不同的作用,直接影响多表位蛋白的构象,进而会增强或抑制多表位蛋白的免疫效应.
목적 연구재체융합태단대인공중조다표위단백M.RCAg-1구상、면역원성급체외보호성적영향.방법 장인공다표위항원M.RCAg-1기인통과불동적매절위점극륭도원핵표체재체pDS-ex상,종이획득대유불동재체융합표첨혹몰유표첨적단백,표체산물경순화득도P312-1、P312-2、P312-3저3충다표위단백,이용생물신식학화색보기술대제비적3충단백적이급급삼급결구진행료분석,병장저3충순도대우95％적중조단백여불씨좌제유화후분별면역접충소서화신서란백토,면역후혈청안상규진행ELISA간접법검측항체적도,채용매련면역반점시험(ELISPOT)검측분비세포인자적특이성림파세포극륭수,동시진행료간접면역형광분석(IFA)이급대체외배양적악성학원충적생장억제시험(GLA).결과 P312-1、P312-2、P312-3저3충다표위단백재동물모형체내유도적항체적도수평병무차이(P＞0.05),단단백면역소서후각유도료불동류형적T세포반응,이차3충단백면역혈청IgG체외생장억제솔출현명현차이(분별위93.9％、14.7％、54.3％).이용생물신식학화색보기술분석,발현종불동표체재체제비적중조단백재이급화삼급결구상출현겁대차이,예측단백질분자적공간구상발생료개변,이차일사표위재분자중적위치야유명현적개변.결론 불동적재체태융합표체대다표위단백M.RCAg-1결구급면역활성적영향회산생불동적작용,직접영향다표위단백적구상,진이회증강혹억제다표위단백적면역효응.
Objective To explore the effects of vector fusion peptides on the conformation and immune reactivity of recombinant polyepitope antigens,M.RCAg-1.Methods We subcloned polyepitope artificial antigen gene,M.RCAg-1,into prokaryotic expression vectors,pDS-ex,that contain different fusion tags at the N-terminus or no any tag by different restriction enzyme cutting site.Three recombinant proteins expressed by these vectors,named P312-1,P312-2,and P312-3,were purified and purity is greater than 95％.Then BALB/c mice were vaccinated with the three proteins formulated with Freund's adjuvant through intranasal.Serum were collected to detect specific antibodies of M.RCAg-1 and individual epitope by ELISA ; natural parasite antigen was recognized by indirect immunofluorescence assay; mouse specific T lymphocyte activation was detected by enzyme-linked immunosorbent spot test (ELISPOT) ; and the growth of Plasmodium falciparum in vitro to evaluate by growth inhibition assay(GIA),and analyze secondary and tertiary structures of recombinant proteins from different expression vectors by bioinformatics and circular dichroism technique.Results The P312-1,P312-2 have almost the same amino acid sequence,and the three proteins have the same immunogenicity in animal models(P＞0.05),however,the different proteins elicited various T-cell responses,the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assaysin vitro ( respectively,93.9％,14.7％,54.3％ ).The significant differences of secondary and tertiary structures were shown in recombinant proteins from different expression vectors,analyzed by bioinformatics and circular dichroism technique,which demonstrated the change of protein molecule spaces conformation,and the obviously change of some epitope locations.Conclusion These results suggest that the expressed polyepitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and,subsequently,the epitope exposure.Thus,these proteins are able to induce both distinct humoral and cellular immune responses in animal models,and they affect the efficacy of immune inhibition against the parasite,the enhancement or suppresses.