尖吻蝮蛇AaHⅣ催化结构域和非催化结构域免疫保护性比较  

Expression,purification and immunoprotection analysis of catalytic domain and non-catalytic domain of AaHIV from Agkistrodon acutus

作　　者：张雷[1] 曹宇亮[1] 刘明华[1] 

机构地区：[1]第三军医大学西南医院急救部,重庆400038

出　　处：《第三军医大学学报》2014年第10期1055-1058,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81071537);重庆市科技攻关计划(CSTC20105026);第三军医大学临床科研课题(2009XLC15)~~

摘　　要：目的表达尖吻蝮蛇出血性P-Ⅲb型金属蛋白酶AaHⅣ催化结构域和非催化结构域,并鉴定其免疫小鼠后抗体的中和保护性。方法采用基因合成的方法合成尖吻蝮蛇P-Ⅲ6型金属蛋白酶AaHⅣ的催化结构域和非催化结构域cDNA,分别重组在质粒pET28a(+)上,转入BL21(DE3)中表达,产物经亲和层析纯化。昆明小鼠随机分为催化结构域蛋白免疫组、非催化结构域蛋白免疫组、AaHⅣ免疫组、PBS免疫组,每组6只[4周龄,体质量(20±2)g,雌雄各半],3次免疫后分离血清,与AaHⅣ体外孵育后行出血中和实验。结果 ELISA显示重组表达的AaHⅣ催化结构域和非催化结构域均具有免疫原性。出血中和实验显示:催化结构域免疫组和非催化结构域免疫组均较PBS免疫组显著减少出血面积(P<0.05);AaHⅣ免疫组与催化结构域免疫组之间无统计学差异(P>0.05),而与非催化结构域免疫组之间存在统计学差异(P<0.05)。结论 AaHⅣ催化结构域免疫小鼠产生的抗体在中和AaHⅣ出血毒性方面效果更为显著。Objective To express the catalytic domain and non-catalytic domain of AaHIV from Agkistrodon acutus, and analyze the immunoprotective effects in mice. Methods The catalytic domain and non-cat- alytic domain cDNA of AaHIV from Agkistrodon acutus were synthesized and cloned into pET28a（ ＋ ） vector, respectively. The recombinant plasmids were transferred into E. coli BL21 （ DE3 ）. Expression products were finally purified by affinity chromatography. Kunming mice were randomly divided into 4 groups （ n = 6, 4 weeks, 20 ± 2 g, male/female ratio = 1 ： 1 ）, including a catalytic domain group, a non-catalytic domain group, an Aa- HIV group and a PBS group. After the third round of immunization, the antiserum was obtained. Titer was determined by ELISA and the protective effect of antiserum was analyzed by hemorrhagic activity test after neutralization of AaHIV. Results Both of the 2 recombinant fragments of AaHIV had immunogenicity. Neutralization test showed that as compared to the PBS group, the catalytic domain group and the non-catalytic domain group both showed significantly reduced subcutaneous hemorrhage （P 〈 0. 05 ）. The subcutaneous hemorrhage area of the AaHIV group was significantly different from that of the non-catalytic domain group （ P 〈 0. 05 ） but not from that of the catalytic domain group （P 〉 0.05 ）. Conclusion The antiserum induced by the catalytic domain of AaHIV from Agkistrodon acutus can more effectively neutralize the hemorrhagic activity of AaHIV.

关 键 词：尖吻蝮蛇 蛇毒金属蛋白酶 SVMPs 催化结构域 非催化结构域 

分 类 号：R345[医药卫生—基础医学] R392.13