论文标题:鹅细小病毒VP基因片段在原核系统中的表达及抗血清的制备
Expression of VP Gene Fragments of Goose Parvovirus in Prokaryotic System and Preparation of Antiserum
论文作者
论文导师 孔宪刚；冉多良；刘胜旺,论文学位 硕士,论文专业 预防兽医学
论文单位 新疆农业大学,点击次数 165,论文页数 56页File Size3399K
2006-06-01论文网 http://www.lw23.com/lunwen_2865307/
Goose parvovirus; structural viral proteins; prokaryotic expression; antiserum
将编码Goose parvovirus(GPV)HG5／82株病毒衣壳蛋白的基因分为三个有部分重合的基因片断，它们分别是从VP1的5’-端到662位核苷酸，从VP2的5’-端到969位核苷酸及从VP3的3’-端到864位核苷酸的基因片段，这三个片段的两个重合区分别包含了VP2-VP3非重合区和VP3的中部，再分别将这三个基因片断连入原核表达系统中进行表达，并将它们相应的表达产物命名为F1、F2、F3。前期工作已经完成了F2的原核表达纯化、抗血清的制备及其免疫活性的检测。 本研究将VP1的5’-端到662位核苷酸和从VP3的3’-端到864位核苷酸的基因片段分别连入原核表达载体pPROEX~(TM)HTb和pET30a中，将构建的重组质粒进行确证性序列测定。再把这两种重组质粒分别转化入大肠杆菌DH5α和BL21，用IPTG诱导后分别表达出与预期大小相符的约32kDa和34kDa的融合蛋白。将它们分别命名为F1和F3，通过薄层扫描分析显示F1表达量最多可占工程菌菌体总蛋白的22.8％。分别诱导两种工程菌后用6mol／L盐酸胍裂解，再经超声处理后离心，用镍离子亲和树脂对裂解产物的上清进行纯化，用于分别制备F1和F3的兔抗血清。Western blot结果表明：F1的抗血清不但能与F1反应还能与亲本株GPV HG5／82的两条蛋白带发生反应，这两条蛋白带的分子量分别为88kDa和77kDa，它们的大小分别与GPV VP1和VP2的相对分子量相同；F3的抗血清不但能与F3反应还能与亲本株GPV HG5／82的四条蛋白带发生反应，这四条蛋白带的分子量分别为88kDa、77kDa、65kDa和60kDa，其中分子量为88kDa、77kDa和60kDa的蛋白带分别与GPV的三种结构蛋白，即VP1、VP2和VP3的分子量一致。该结果证明融合蛋白F1和F3与F2一样具有良好的免疫原性。由于GPV三种结构蛋白具有共同的羧基端，因此在理论上，存在于VP3的表位应该也存在于VP1和VP2；存在于VP2的抗原表位应该也存在于VP1。通过分析F1、F2和F3的兔抗血清与亲本株的Western blot结果，初步对GPV HG5／82株VP蛋白的B细胞线性抗原表位进行定位：VP1的第145～198位和220～733位氨基酸残基中含有B细胞线性抗原表位，第198～220位氨基酸残基之间没有B细胞线性抗原表位。 本研究为定位GPV VP蛋白的抗原表位及开发研制抗鹅细小病毒感染的基因工程疫苗和诊断试剂提供依据。
In order to mapping B cell sequencial epitopes and study the antigenicity of viral structural protein of goose parvovirus(GPV), we put the vp gene into 3 fragments with overlapped vp2-vp3 and the middle of vp3. They are 662bp at 5"-end of vp1, 969bp at 5"-end of vp2, and 864bp at 3"-end of vp3. We planed to express these fragments in prokaryotic expression system to study the reactivity of these fusion protein, and tentatively named these expresssed fusion protein as F1、 F2、 F3, respectively. In previous study , F2 had been expressed and purified. The antiserum against F2 had been prepared by immunizing rabbit, and reactivity of the antiserum had been testified by western blot.In this study, a 662bp fragment at 5"- end of vp\ gene and a 864bp fragment at 3"- end of vp gene of GPV isolate HG5/82 was amplified by PCR. We inserted the 662bp fragment at 5"- end of vp\ gene between BarnH I and Hind III site of prokaryotic expression vector pPROEX~(?)HTb and put the 864bp fragment at 3"- end of vp gene into the same restriction endonuclease site of prokaryotic expression vector pET30a, respectively. After identification of recombinant plasmids by sequencing, the recombinant pPROEX~(?)HTb was transformed into Escherichia coli DH5a and the recombinant pET30a was transformed into Escherichia coli BL21. SDS-PAGE results indicated that transformed Escherichia coli DH5a expressed a 32kDa product and the transformed Escherichia coli BL21 expressed a 34kDa product after inducing with 0.6mmol/L IPTG. Densitometric scanning showed the expressed F1 accounted for as much as 22.8% of total bacterial protein of DH5 α, it indicated that the target gene can be expressed in this prokaryotic expression system in high efficiency. We named the 32kDa product as F1 and the 34kDa product as F3, respectively. As the fusion proteins have 6 x His tag, F1 and F3 were purified with a high purity by ProBond~(?) Resin. By immunizing rabbits the antiserum against F1 and F3 were obtained. Western blot analysis indicated that both of these two kinds of antiserum could recognize the GPV HG5/82 stain"s protein, but they identified different viral structural proteins. Antiserum against F1 recognised VP1 and VP2, and antiserum against F1 identified VP1、 VP2 and VP3 of GPV HG5/82. The three kinds of structural proteins have identical carboxyl terminal, so the epitope on the VP3 also exists on the VP1 and VP2, while the epitopes on the VP2 are also present on the VP1. Accoding the theory and results of western blot, B cell linear epitopes were mapped in amino acids at 145-198 and amino acids at 220-733 of VP1 sequence of GPV HG5/82 strain. These is no B cell linear epitopes at 198-220 amino acids of VP1 sequence of GPVHG5/82 strain.These data provide more useful information for antigenic epitope prediction of .GPV capsid protein. It established a foundation and prepared experimental material for the future research on the bioactivity of GPV, and also provided a basis for developing GPV recombiant diagnosis reagent and genetic engineering vaccine.
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