【作者】 王志宏；

【导师】 郭存九；

【作者基本信息】 山西医科大学 ， 临床检验诊断学， 2014， 硕士

【摘要】 目的：柯萨奇病毒A组16型(CA16)和肠道病毒71型(EV71)是手足口病(HFMD)的两个主要致病原。目前的研究发现,HFMD爆发时,CA16和EV71常常交替或共同流行。然而,由于CA16反复感染以及与其他肠病毒的交叉反应性,使得CA16体液免疫反应复杂,CA16体液免疫反应特点尚不明确。本实验构建CA16VP1全长及各截短抗原,利用NEIBM的ELISE方法,研究柯萨奇病毒A组16型体液免疫反应特点。方法：构建pET21b-CA16VP11-297aa(全长)、pET32a-CA16VP141-297aa/pPEPTIDE2-CA16VP11-60aa、 pPEPTIDE2-CA16VP145-58aa和pET32a-CA16VP161-297aa原核表达质粒,转入大肠杆菌经IPTG表达,Ni亲和柱层析纯化上述重组蛋白,ELISA鉴定CA16VP1全长重组蛋白,用NEIBM间接ELISA方法检测95份太原市老年人群(平均年龄：61.0±14.0岁)及142份上海市献血员人群(平均年龄：35.5±11.2岁)的血清抗CA16VP1的抗体情况,并进行竞争ELISA分析。结果：1.成功构建了5种CA16VP1全长及截断重组质粒并在大肠杆菌中表达,获得了5种VPl全长及截断重组蛋白,其中pPEPTIDE2-CA16VP11-60, pPEPTIDE2-CA16VP145-58以可溶性蛋白形式存在,pET32a-CA16VP161-297, pET32a-CA16VP141-297, pET21b-CA16VP11-297以包涵体的形式存在。2. NEIBM间接ELISA检测结果显示：太原市老年人群血清与CA16VP11-297、CA16VP141-297. CA16VP11-60. CA16VP145-58和CA16VP161-297抗原反应的阳性率分别为76.84%、40.00%、33.68%、31.57%和30.53%；上海市献血员人群血清抗-CA16VP11-297的阳性率为59.15%。3.对46份太原市老年人抗-CA16VPl1-297强反应血清的竞争ELISA结果显示：CA16VP11-297、CA16VP141-297、 CA16VP11-60、 CA16VP145-58和CA16VP161-297抗原的抑制率中位数分别为73.50%、53.75%、21.00%、20.50%和15.50%。对84份上海市献血员抗CA16VP11-297强反应血清的竞争ELISA结果显示：CA16VP11-297、 CA16VP141-297、 CA16VP11-60、 CA16VP145-58和CA16VP161-297抗原的抑制率中位数分别为86.00%、86.00%、82.50%、62.00%和28.50%。4.太原老年人血清和上海献血员血清与CA16VPl全长及各截短蛋白的竞争模式及构成比明显不同。结论：1.太原老年人群及上海献血员人群中有较高的CA16和/或CA16相关抗体。2CA16VPl全长及各截短抗原对太原老年人群和上海献血员人群中抗-CA16VP1检测的抑制情况明显不同,提示这两类人群感染了不同的CA16和/或CA16相关病毒。3.人体抗CA16抗体反应主要针对以交叉表位为主的抗原。更多还原

【Abstract】 Objetive:Coxsackie virus type16group A (CA16) and enterovirus71(EV71) are two major pathogenic agents of hand, foot and mouth disease (HFMD). The current study found that When HFMD outbreak, EV71and CA16often alternate or popular together. However, due to the CA16infection and repeatedly cross reactivity with other enteroviruses, makes the CA16complex, humoral immune response characteristics of CA16is unclear. This study to constrct CA16VP1full-length and the truncated antigen, using NEIBM ELISE method, study on the characteristics of human body against coxsackievirus A16humoral immune response.Method:Prokaryotic expression plasmid pET21b-CA16VP11-297aa, pET32a-CA16VP141-297aa, pPEPTIDE2-CA16VP11-60aa, pPEPTIDE2-CA16VP145-58aa and pET32a-CA16VP161-297aa were constructed, transformed into E.coli BL21(DE3) for expession under induction of IPTG, The expressed recombinant proteins were purified by nickel ion affinity chromatography, The expressed recombinant proteins CA16VP11-297was identified by ELISA,using NEIBM indirect ELISA method to detect anti CA16VP1antibodies with95serums of the elderly in taiyuan city(mean age:61.0±14.0years) and142serums of blood donation members in Shanghai city (average age:35.5±11.2years), and to analysis by competitive ELISA.Result:1.Successfully constructed five kinds of CA16VP1the complete and truncated recombinant plasmid and expressed in escherichia coli, obtained the five kinds of VP1and truncated recombinant proteins, including pPEPTIDE2-CA16VP11-60, pPEPTIDE2-CA16VP145-58exists in the form of soluble protein, pET32a-CA16VP161-297,pET32a-CA16VP141-297,pET21b-CA16VP11-297exists in the form of inclusion body.2. NEIBM indirect ELISA test results showed that: positive rate of reaction of taiyuan elderly serums with CA16VP11-297, CA16VP141-297, CA16VP11-60, CA16VP145-58and CA16VPl61-297antigens were76.84%,40.00%,33.68%,31.57%and30.53%; Shanghai blood donation member crowd serums anti-CA16VP11-297positive rate was59.15%..3. Competitive ELISA results of Anti-CA16VP11-297strong reaction serums of46taiyuan older showed that:CA16VP11-297, CA16VP141-297, the CA16VP11to60, CA16VP145-58and CA16VP161-297antigen inhibition rate median were73.50%,53.75%,21.00%, 20.50%and15.50%. Competitive ELISA results of Anti-CA16VP11-297strong reaction serums of84Shanghai blood donation members showed that:CA16VP11-297, CA16VP141-297, the CA16VP11-60, CA16VP145-58and CA16VP161-297antigen inhibition rate median were86.00%、86.00%、82.50%、62.00%and28.50%.4. The competition patterns and composition rario of taiyuan elderly serums and Shanghai blood donation members serums were obviously different.Conclusion:1.The elderly crowd in Taiyuan city and the adult population in Shanghai city have higher CA16and/or CA16related antibodies.2. Testing inhibition situation of CA16VP1the full-length and the truncated antigen with anti-CA16VP1of taiyuan elderly and Shanghai crowd are obviously different, suggest the two types of people infected with different CA16and/or CA16related viruses.3. Human anti-CA16antibody reaction mainly focusing on cross epitope based antigen.更多还原