【作者】 卢爱桃；

【导师】 呼和巴特尔； 吕亦晨；

【作者基本信息】 内蒙古农业大学 ， 预防兽医学， 2008， 博士

【摘要】 猴T淋巴细胞白血病毒I型(STLV-1)、猴单纯疱疹病毒(BV)在国内猴群中的感染率比较高,这不但影响动物健康和实验研究的结果,而且对实验接触人员具有严重危害性。目前,分离、细胞培养病毒作为抗原是诊断这两种疾病常用的方法,这对实验人员有严重的危害性,而且成本相对较高。为了获得更多的、对实验操作人员没有危害的抗原,本研究采用了体外化学合成猴T淋巴细胞白血病毒I型(STLV-1)gag、单纯疱疹病毒(BV)gd部分保守基因片段,经大肠杆菌的表达、亲和层析纯化,建立了STLV-1、BV的ELISA诊断方法,同时,建立了一种可以同时检测两种病毒抗体的液相芯片技术,这为以后的进一步实验奠定了基础。本研究根据Genbank公布序列化学合成STLV-1gag、BV gd基因片段,经酶切后将目的片段连接到表达载体pET15b中并进行测序鉴定。再将重组质粒转化到表达宿主菌BL21(DE3)中,经小量优化诱导表达条件后进行SDS-PAGE、Western Blot检测表达量及表达活性,经蛋白质活性鉴定及大小均正确的菌株进行大量(10L)发酵、纯化得到重组蛋白STLV-1 gag和重组蛋白BV gd。然后以重组蛋白作为抗原检测猴血清中相应抗体,并与现有商品化诊断试剂盒结果对比。同时,将重组蛋白STLV-1 gag、重组蛋白BV gd作为探针偶联到不同颜色(即不同编号)的荧光微球上对恒河猴血清中相应的抗体进行检测,检测结果与ELISA检测结果进行对比。经研究得到如下结果:1.从化学合成的pJ-STLV-1 gag、pJ-BV gd载体经酶切获得了目的片段STLV-1 gag (1323bp), BV gd (1194bp),然后将目的片段与表达载体pET15b连接,构建了重组表达质粒pET15b-STLV-1 gag、pET15b-BV gd。2.经鉴定正确的重组质粒转入表达宿主rosetta2 DE3 plysS中,小量诱导表达STLV-1 gag、BV gd,优化诱导表达条件后进行SDS-PAGE、Western Blot检测表达量及表达活性。结果表明,重组蛋白STLV-1 gag、重组蛋白BV gd在大肠杆菌中成功表达,STLV-1 gag重组蛋白以可溶形式存在于菌体中,BV gd重组蛋白以包涵体的形式存在。3.10L发酵罐大量诱导大肠杆菌表达重组蛋白STLV-1 gag和重组蛋白BV gd,然后经AKTA纯化系统Ni2+亲和层析纯化得到重组蛋白STLV-1 gag和重组蛋白BV gd。4.以重组蛋白STLV-1 gag为抗原检测广西某猴场1304份恒河猴血清,阳性率为3.76%,重组蛋白BV gd作为抗原检测514份恒河猴血清,阳性率为29.96%;现有商品化诊断试剂盒检出阳性率分别为2.68%、21.60%。经Western Blot确认检测结果,重组蛋白检出率明显高于现有商品化诊断试剂盒(P<0.05)。因此重组蛋白STLV-1 gag和重组蛋白BV gd可以作为病毒良好的代替抗原,解决了通过分离、培养病毒获取抗原的困难以及操作的危险性。同时,为猴群的净化、SPF实验用猴的保证提供了一种廉价、快速、准确、有效的ELISA诊断方法。5.对比ELISA与流式荧光微球检测结果,证明用荧光微球对猴血清进行检测,不仅比ELISA法快速省时,而且灵敏度比间接ELISA法高出5～100倍。建立了一种更为灵敏、方便快速、高通量的荧光微球诊断技术,在一个反应体系中可以同时检测多个指标,这与传统方法的逐个检测相比是一个质的飞跃。更多还原

【Abstract】 The infection rate of Simian T-Lymphotropic/Leukemia Virus type I (STLV-1) and Herpesvirus simiae (BV) in China monkey is very high. It not only affected the animal health and experiment results, but also was harmful to operators. At present, the diagnosis method of the two diseases are separating and cell culturing virus as antigen, it is harmful to operators and more expensive. In order to obtain more and harmless antigen, this study used Simian T-Lymphotropic/Leukemia Virus type I (STLV-1) gag chemical synthesis, Herpesvirus simiae (BV) gD gene which is part of the conservative fragment, then expressed it in E.coli, purified it by affinity chromatography, and established a STLV-1, BV of ELISA diagnostic method, at the same time, established a liquid chip technology about detecting two viruses antibodies together, This will lay the foundation for further experiments.We synthesized of STLV-1gag, BV gd gene fragment according to the sequence published on Genbank. Linked the purpose fragments into expression plasmid pET15b after digestion and was sequenced. It was transformed into the expression host of BL21 (DE3).After induced optimization conditions is confirmed, we detected the amount and activity of expression by SDS-PAGE and WB. Then operated fermentation (10 L) and purification of recombinant protein STLV-1 gag, BV gd. Recombinant protein used as antigen to detect the corresponding antibody in monkey serum, and compared with commercialization of diagnostic kit results. At the same time, recombinant protein STLV-1 gag, BV gd as a probe coupled to different colors (different #) fluorescent microbeads to detect the corresponding antibodies in rhesus monkey serum, and compared the test results with ELISA test results. The results are as follows:1. Got sequence fragments STLV-1 gag (1323bp), BV gd (1194bp) from pJ vector which is chemical synthesis by DNA2.0 Inc, and linked purpose fragment with the expression vector pET15b,so recombinant expression plasmid pET15b-STLV-1 gag, pET15b-BV gd had been constructed.2. Transformed the plasmid into the expression host rosetta2 DE3 plysS which was identified correctly. Detected the amount and expression of activity by SDS-PAGE, Western Blot after induced optimization conditions was confirmed. The results showed that STLV-1 gag, BV gd recombinant protein were expressed successfully in the Escherichia coli, STLV-1 gag recombinant protein existed bacteria in the form of soluble, BV gd recombinant protein in the form of inclusion bodies.3. Induced recombinant protein expression in 10L fermenter and purified recombinant protein STLV-1 gag, BV gd by Ni2+affinity chromatography in AKTA purification system.4. Used STLV-1 gag recombinant protein as antigen for detecting 1304 monkey sera from Guangxi, the positive rate was 3.76%; used BV gd recombinant protein as antigen for detecting 514 monkey sera, the positive rate was 29.96%; the positive rates respectively 2.68%, 21.60% by commercial diagnostic kits. The results showed that detection rate of recombinant protein significantly higher than commercial diagnostic kit (P <0.05). Therefore STLV-1 gag, BV gd can be used as a good substitute virus antigen. This will eliminate the dangers and difficulty of separating and culturing virus. At the same time, this will provide a cheap, rapid, accurate and effective ELISA method which can help to purify SPF monkey.5. Compared to ELISA and fluorescent microbeads flow test results, it proved that the fluorescent microbeads detecting monkey serum not only was more rapid than ELISA method, but also more sensitive and high than indirect ELISA method. It’s established a more sensitive, convenient and rapid fluorescent microbeads diagnostic technique which can detect multiple targets simultaneously in a reaction system. It is a qualitative leap compared to the traditional methods of detection.更多还原