猪圆环病毒Ⅱ型Cap蛋白噬菌体单链抗体库的构建与筛选

发布时间：2018-05-16 03:23

本文选题：PCV2 + Cap蛋白　；参考：《郑州大学》2017年硕士论文

【摘要】：猪圆环病毒Ⅱ型(Porcine circovirus,PCV2)是引起猪圆环病毒病(Porcine circovirus disease,PCVD)的主要病原,它能损害患病仔猪的免疫系统,引发其他多种病原混合感染或继发性感染,导致猪的繁殖能力严重下降,给养猪业带来了巨大的经济损失。PCV2感染的早期诊断对PCVD的综合防控具有重大意义。目前,针对PCV2病原的快速检测方法主要有胶体金免疫层析试纸法和ELISA检测方法等,其所用抗体主要是基于杂交瘤细胞制备的单克隆抗体,传统单克隆抗体存在生产周期长、成本高、稳定性差等缺点,在一定程度上限制了PCV2快速、高效、廉价的检测方法的研制。而基因工程方法制备的单链抗体具有生产周期短,成本低、易于改造等优点,其有望取代传统单抗用于PCV2新型免疫学检测方法的研制。实验的主要内容与结果如下:1.PCV2 Cap蛋白的纯化及鉴定本实验将重组表达载体pET28a-ORF2转入大肠杆菌BL21中,用IPTG诱导表达,利用Ni-NTA亲和层析法对目的蛋白进行纯化,并对蛋白进行鉴定。结果显示表达的重组蛋白分子量约26 kDa,主要以可溶形式存在,且能被His单克隆抗体特异性识别,纯化后的重组蛋白纯度比较高。用此蛋白免疫BALB/C鼠,免疫后,经ELISA检测,测得鼠血清中抗体滴度达1∶64000,同时用PCV2抗体检测试纸对鼠血清进行检测,结果为阳性。本实验制备的重组Cap蛋白具有可溶性好、纯度高、免疫原性好等特点,为Cap蛋白单链抗体库的构建提供了必要的实验材料。2.PCV2 Cap蛋白噬菌体单链抗体库的构建及淘选选取免疫效果较好的小鼠脾脏,提取其脾脏总RNA,再以反转录后的cDNA为模板,PCR扩增抗体的重链可变区基因(Heavy chain variable region of antibody,VH)和抗体的轻链可变区基因(Light chain variable region of antibody,VL),经重叠PCR将VH与VL用多肽(G4S)3连接,获得单链抗体基因(Single chain antibody variable region gene fragment,ScFv)。经辅助噬菌体辅助侵染后获得噬菌体单链抗体库,用Cap蛋白作为包被原筛选PCV2单链抗体克隆,获得3株与Cap蛋白特异性反应的噬菌体克隆。取特异性及反应性相对较高的克隆测序,获得PCV2单链抗体基因,并用ELISA鉴定ScFv-P18菌株的诱导表达后粗提物,结果显示获得的粗提物能与Cap蛋白发生反应。本研究表达并纯化到了可溶性PCV2 Cap蛋白,并构建PCV2 Cap蛋白噬菌体单链抗体库,筛选到具有结合活性的特异性单链抗体,其有望用于PCV2新型快速检测方法的研制。
[Abstract]:Porcine circovirus type 2 (Porcine circovirus) is the main pathogen causing porcine circovirus disease (PCVD2). It can damage the immune system of infected piglets and lead to mixed or secondary infection of many other pathogens, leading to a serious decline in the reproductive ability of pigs. The early diagnosis of PCV2 infection is of great significance to the comprehensive prevention and control of PCVD. At present, the main methods for rapid detection of PCV2 pathogens are colloidal gold immunochromatographic test paper method and ELISA detection method. The antibodies used are mainly based on monoclonal antibodies prepared by hybridoma cells, and the traditional monoclonal antibodies have long production cycle. The disadvantages of high cost and poor stability limit the development of fast, efficient and cheap detection method for PCV2. However, the single-chain antibody prepared by genetic engineering method has the advantages of short production cycle, low cost and easy modification. It is expected to replace the traditional monoclonal antibody for the development of a new immunological detection method for PCV2. The main contents and results are as follows: 1. Purification and identification of PCV2 Cap protein. In this experiment, the recombinant expression vector pET28a-ORF2 was transferred into Escherichia coli BL21 and expressed by IPTG. The target protein was purified by Ni-NTA affinity chromatography, and the protein was identified. The results showed that the molecular weight of the expressed recombinant protein was about 26 kDa, which existed mainly in soluble form and could be specifically recognized by His monoclonal antibody. The purified recombinant protein was of high purity. BALB/C mice were immunized with this protein. The titer of antibody in the serum of mice was 1: 64000 by ELISA detection. The serum of mice was detected with PCV2 antibody test paper and the results were positive. The recombinant Cap protein prepared in this experiment has the characteristics of good solubility, high purity and good immunogenicity. The necessary materials for the construction of Cap single chain antibody library were provided. 2. The construction of phage single chain antibody library of PCV2 Cap protein and the selection of mouse spleen with better immune effect by panning. The spleen total RNAs were extracted, and the heavy chain variable region of antibody VHs were amplified by reverse transcription cDNA template. The heavy chain variable region of antibody VHs and the light chain variable region of antibody VHs were amplified by overlapping PCR. VH and VL were linked with polypeptide G4Sf3 by overlapping PCR. Single chain antibody variable region gene fragment was obtained. The phage scFv library was obtained after assisted phage infection. PCV2 scFv clones were screened with Cap protein as the coating, and three phage clones specifically reacted with Cap protein were obtained. The PCV2 single chain antibody gene was obtained by cloning and sequencing, and the crude extract of ScFv-P18 strain was identified by ELISA. The results showed that the obtained crude extract could react with Cap protein. In this study, soluble PCV2 Cap protein was expressed and purified, and PCV2 Cap phage scFv library was constructed, and specific scFv with binding activity was screened, which is expected to be used in the development of a new rapid detection method for PCV2.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

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