副溶血弧菌基因芯片及基因工程单链抗体检测研究

发布时间：2018-12-14 21:55

【摘要】：副溶血弧菌是一种广泛存在于水环境中的一种导致人类和水生动物发生疾病的一种致病菌。在本研究中,我们建立了一种基于基因芯片和基于基因工程单链抗体的融合蛋白的检测方法,对副溶血弧菌进行检测。以副溶血弧菌的基因组的特征性基因为出发点,利用副溶血弧菌不耐热溶血毒素基因(tlh)上的367 bp的片段检测TLH,耐热溶血毒素基因(tdh)上269 bp的片段检测TDH,鞭毛蛋白编码基因(FlaB)上的176 bp的片段检测FLAB,利用Bio-dUTP标记的液相三重PCR产物与特异探针(引物)的杂交,结合碱性磷酸酶检测系统,应用基因芯片技术同步检测副溶血弧菌的三个特有基因。实验中,使用了36份的实际样品对该检测体系作了进一步的验证分析,在检测的18株副溶血弧菌中,检测到有8株是强毒株,10株是弱毒株,这与实际的结果相符合。在检测的18株其他细菌中,均未发现任何的非特异性条带,这与实际的结果也是一致的。通过PCR扩增副溶血弧菌基因组中的约1.3 kb的tlh抗原基因,利用基因工程技术,构建了pET32a-tlh载体,将抗原基因与硫氧还原蛋白基因进行融合,转化大肠杆菌并进行IPTG诱导表达。SDS-PAGE凝胶电泳分析表明有一条与预测分子量大小相一致的目的条带,表达量约占菌体蛋白的45%左右,蛋白的可溶性高。融合蛋白末端的Trx标签增加蛋白的可溶性,His6标签有利于蛋白质的纯化。用纯化后的蛋白免疫Balb/c小鼠,经过加强免疫后测定小鼠的抗血清效价,达到一定的效价后,从小鼠的脾脏中提取总RNA,反转录得到cDNA第一链,通过PCR扩增得到重链可变区V_H和轻链可变区V_L基因,组装成scFv并克隆到噬菌体载体pCANTAB-5E中,构建噬菌体抗体库。通过噬菌体展示技术淘选得到一株高亲和力的单链抗体WW6。
[Abstract]:Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a pathogen that causes diseases in human and aquatic animals. In this study, we developed a method for detection of vibrio parahaemolyticus based on gene chip and genetic engineering scFv. Based on the characteristic genes of the genome of Vibrio parahaemolyticus, the 367 bp fragment of the thermostable hemolytic toxin gene (tlh) of Vibrio parahaemolyticus was used to detect the 269 bp fragment of the TLH, heat resistant hemolytic toxin gene (tdh) for TDH, detection. Detection of 176 bp fragment on flagellin encoding gene (FlaB) FLAB, used Bio-dUTP labeled liquid phase triple PCR products hybridization with specific probes (primers), combined with alkaline phosphatase detection system. Three unique genes of Vibrio parahaemolyticus were detected by gene chip technique. In the experiment, 36 actual samples were used to further verify and analyze the detection system. Of the 18 strains of Vibrio parahaemolyticus detected, 8 were virulent strains and 10 were attenuated strains, which was consistent with the actual results. No nonspecific bands were found in the 18 other bacteria tested, which is consistent with the actual results. About 1.3 kb tlh antigen gene in the genome of Vibrio parahaemolyticus was amplified by PCR. The pET32a-tlh vector was constructed by genetic engineering technique, and the antigenic gene was fused with thioxy-reducing protein gene. SDS-PAGE gel electrophoresis analysis showed that there was a target band which was consistent with the predicted molecular weight of E. coli. The expression level was about 45% of the bacterial protein and the soluble protein was high. The Trx tag at the end of the fusion protein increased the solubility of the protein, and the His6 tag was helpful to the purification of the protein. Balb/c mice were immunized with purified protein. The titer of antiserum was determined after enhanced immunization, and the total RNA, reverse transcriptase was extracted from the spleen of the mice to obtain the first chain of cDNA. The V _ (th) gene in the heavy chain variable region and the V _ (L) L gene in the light chain variable region were obtained by PCR amplification. The scFv was assembled and cloned into the phage display vector pCANTAB-5E to construct the phage antibody library. High affinity scFv WW6. obtained by phage display technique
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.1

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