【作者】 李润成；

【导师】 余兴龙；

【作者基本信息】 湖南农业大学 ， 预防兽医， 2007， 硕士

【摘要】 1、三种猪O型口蹄疫抗体检测试剂盒的比较应用两种ELISA试剂盒(赛迪公司FMDV O型抗体检测阻断ELISA试剂盒，兰州所FMDV O型抗体检测液相阻断ELISA试剂盒)和一种正向间接血凝试剂盒(兰州所生产的FMDV O型正向间接血凝抗原试剂盒)对125份口蹄疫免疫猪血清进行了抗体水平测定，并对三种试剂盒测定结果的符合程度、及其各自的重复性进行了分析。试验结果表明三种试剂盒测得的抗体水平之间有较好的一致性，且三种试剂盒各自的检测结果重复性良好。这一工作为FMDV基因工程疫苗动物免疫试验的免疫学检测确定了一种可靠的方法。2、O型口蹄疫病毒代表性VP1基因的人工合成、表达及重组蛋白的免疫原性研究通过对GeneBank近十年内收录的O型口蹄疫病毒VP1基因序列的同源性比较分析，根据与我国口蹄疫防制工作有密切相关性的一个基因群的基因序列设计并合成了有群内抗原代表性的结构蛋白VP1全基因片段。将合成的序列克隆到原核表达载体pET-28a(+)中、构建了VP1的表达质粒pETVP1。SDS-PAGE和Western-blot分析表明该质粒的BL21(DE3)LysS转化菌在IPTG诱导下可特异性表达VP1蛋白，该重组蛋白以包涵体的形式存在、分子量约30KD，且能与口蹄疫病毒(FMDV)阳性血清发生特异性反应。动物接种实验表明重组蛋白能诱导小白鼠产生特异抗FMDV抗体。3、O型FMDV VP1基因与大肠杆菌不耐热肠毒素B亚单位(LTB)基因的融合表达及表达产物的免疫原性研究以质粒pMDTLT为模板、用PCR的方法扩增出LTB基因，然后将其插入到pETVP1质粒中VP1基因的上游、构建了含有融合基因LTBVP1的表达质粒pETLTBVP1，转化宿主菌BL21(DE3)LysS后进行诱导表达。表达产物经SDS-PAGE显示、重组蛋白以包涵体的形式表达、分子量约39KD；经Western-blot分析表明重组蛋白能与FMDV阳性血清及兔抗霍乱毒素(CT)血清反应，说明融合蛋白保持了LTB和VP1各自的免疫学活性。动物实验表明：该融合蛋白能诱导小鼠产生较强的免疫应答反应，其水平比试验中选用的普通商品口蹄疫苗所诱导的抗体水平还要高。4、LTB对VP1蛋白粘膜免疫效应增强作用的研究本实验将前面2个实验中的VP1、LTBVP1重组蛋白纯化后分别通过滴鼻、灌胃、灌肠等粘膜途径接种小鼠；最后采用正向接间血凝检测小鼠血清抗体水平，结果表明：单纯的VP1重组蛋白通过粘膜途径多次接种后能诱导出较弱的免疫反应，而LTBVP1融合蛋白二免后既能诱导小鼠产生较高水平FMDV特异性抗体。说明LTB可增强VP1蛋白通过粘膜途径所诱导的免疫反应。更多还原

【Abstract】 1、Comparison of Three Assays for detection of antibodiesagainst Foot-and-Mouth Disease Virus Serotype OAntibody level of 125 sera from farms were detected by two ELISA and a IHAassays for detection of antibodies against Foot-and-Mouth Disease Virus Serotype O.The coincidence rate and repeatability of three kits were analyzed o The results indictthat antibody level determined by three kits showed good accordance. The resultsdetected by three kits repeat well.2、Synthesis, expression of representative VP1 gene of FMDVtype O and detection of expression product immunogenicityThe VP1 gene of FMDV type O was designed and synthesized artificiallyaccording to gene sequences of FMDV genetic group 1 that is close relative to diseasecontrol in our country by homology comparison among FMDV oligonucleotidesequence that were published by GeneBank in the past ten years. And the expressionplasmid carrying VP1 gene was obtained by the technology of genetic engineering.The expression vector was transformed into BL21 (DE3)LysS. VP1 was expressed inBL21(DE3)lyss and was analyzed by SDS-PAGE and Western-blot. The resultshowed that the recombinant protein found in inclusion bodieso It had a molecularweight of approximately 30 KD, and the protein was able to react with FMDVpositive serum. The protein was used to vaccinate mice, whose immune responseswere then observed. The protein could induce serum antibody. 3、Expression and immunogenesity analysis of a recombinantfusion protein of LTB and VP1LTB gene fragment was amplified by PCR from plasmid PMDLT, Arecombinant expression plasmid pETLTBVP1 was constructed by inserting LTB genefragment into VP1 gene expression vector pETVP1that was constructed in the secondexperiment. The recombinant expression plasmid pETLTBVP1 was transformed toBL21(DE3)LysS for expression. Identify the expressed product by SDS-PAGE andanalyze its immunoreactivity by Western blot. The results showed the molecularweight of fusion protein was about 39 KD, and the recombinant protein was found ininclusion bodies; The fusion protein was able to react with FMDV positive serum andanti-CT serum of rabbit. Animal experiment result showed that, compared withconventional inactivated vaccines, this fusion protein elicited higher antibody levelsof serum antibody.4、Enhancing Effect of B Subunit of Escherichia coli Heat-labileEnterotoxin on Mucosal Immunity of FMDV VP1 ProteinThe VP1 protein of FMDV type o and fusion protein of VP1 with LTB wereobtain under IPTG induction. The expressed proteins were purified, and Mice wereinoculated with purified proteins VP1 proteins and LTB-VP1 fusion protein by directnasal drops, stomach delivery and rectal instillation respectively, and the antibodylevels of Mice was detected. The results showed that the anti-FMDV antibody in seraof mice immunized with LTB-VP1 fusion protein was higher than mice immunizedwith VP1protein in all of the immunity groups.更多还原