【作者】 贾丽锋；

【导师】 曹刚强； 王云龙；

【作者基本信息】 郑州大学 ， 遗传学， 2010， 硕士

【摘要】 流感病毒是流行性感冒的病原体,根据其核蛋白与基质蛋白抗原性的不同分为甲,乙,丙三型。A型流感病毒(Influenza A virus)属正粘病毒科,由8个节段组成的单股负义RNA链,共编码10种多肽,其中由第5节段的RNA编码的核蛋白(Nucleoprotein, NP)高度保守,具有种群和型的特异性,是流感病毒分型和诊断的基础。因此,在原核或真核表达系统中表达NP蛋白,可用于A型流感酶免检测方法的探索,为A型流感病毒的临床诊断提供及时准确的参考信息。本研究采用合成的A型流感病毒(A/duck/Zhejiang/11/2000(H5N1))NP DNA基因作为模板,PCR法扩增得NP基因全长为1498bp,编码498个氨基酸,构建重组质粒并转化表达载体,所表达的A型流感NP蛋白相对分子质量为57 KD左右；将流感NP蛋白免疫动物制备两种抗血清,纯化抗血清中的抗体用作A型流感酶免检测中的包被和标记抗体,最终用于A型流感双抗体夹心检测方法初步探索。本文研究的主要内容：(1)本研究利用人工合成A型流感病毒株H5N1 NP DNA基因序列作为模板,构建原核重组质粒pET-30 Xa/LIC-NP,并诱导该原核重组质粒在大肠杆菌表达系统中表达、纯化A型流感NP蛋白；(2)用纯化后的NP蛋白免疫羊、兔,收取羊抗NP血清、兔抗NP血清,纯化并用HRP标记羊抗NP多抗和兔抗NP多抗；(3)A型流感双抗体夹心检测方法(酶联免疫法)初步探索,使用所制备的多克隆抗体均可作为双抗体夹心中的包被及酶标抗体,对A型流感双抗体夹心检测方法进行可行性分析。本研究采用原核表达系统表达并纯化A型流感NP蛋白,消除了采用天然抗原的生物安全性隐患,是A型流感病毒双抗体夹心ELISA检测方法的初步探索,为开发流感早期诊断试剂盒提供支持。更多还原

【Abstract】 Epidemdic influenza pathogens were influenza viruses, which were divided into A, B, C three types according to their nucleoprotein and matrix protein antigen. Influenza A virus belonged to Orthomyxoviruses Division, forming by eight segments of single stranded negative sense RNA chain, and encoding a total of ten polypeptides. The fifth paragraph of the RNA encoded nuclearprotein (Nucleoprotein, NP),which were highly conserved with the population and type of specificity in the influenza virus,were the basis of classification and diagnosis. Therefore, NP protein expressed in prokaryotic or eukaryotic expression system could be used for detecting of influenza A virus by enzyme immunoassay and seting up a method of explorating for the clinical diagnosis of influenza A virus to provide timely and accurate reference information.In this study, the synthetic NP DNA gene of the influenza A virus (A/duck/Zhejiang/11/2000 (H5N1)) were used as the PCR template.Then,the NP gene, which had a length of 1498 bp and encoded 498 aminoacids, were amplificated by PCR technique. And the recombinant plasmid were constructed and transformed into the expression vector. The relative molecular mass of the expressed influenza A virus NP protein were about 57 KD, and were used to immune the animals and prepare two types of anti-serum and purify the antibodies of the anti-serum, which would be used in the enzyme immunoassay for the detection of influenza A virus. In the end, the marked and packaged antibodies were used in the final double-antibody sandwich method for the preliminary exploration of detecting the influenza A virus.In this paper,the main contents:(1) In this study, the prokaryotic recombinant plasmid pET-30 Xa/LIC-NP was contracted by the template,which were the DNA gene sequences of influenza A virus H5N1 NP strain.And the nucleoprotein were induced in the prokaryotic recombinant plasmid,expressed and purificated in E. coli expression system;(2) Used the purified NP protein to immune the sheep and rabbit to get and purify the anti-NP charge anti-serum, and labeled the goat anti-NP polyclonal antibody and rabbit anti-NP polyclonal antibody with the enzyme of HRP; (3) The initial exploration of influenza A double-antibody sandwich detection method (ELIS A method), used of polyclonal antibodies can be prepared as a double-antibody sandwich of packaged and marked antibodies,which were used for analysising the feasibility of double-antibody sandwich detection method of influenza A virus.In this study, influenza NP protein antigen were expressed and purified in prokaryotic expression system,according to this method, which eliminated the natural bio-safety risks of the influenza virus.The initial exploration of double-antibody sandwich ELISA diagnosis method, providing support to the development of early diagnosis kits of influenza A virus.更多还原