抗H1亚型猪流感病毒单克隆抗体的制备及重链可变区基因的克隆

发布时间：2018-04-21 14:05

本文选题：单克隆抗体 + H1N1猪流感　；参考：《东北农业大学》2009年硕士论文

【摘要】： 用RT-PCR方法从病毒中获得HA基因,构建真核表达质粒HA-pCI-neo。以真核表达质粒HA-pCI-neo肌肉免疫6-8w雌性BALB/c小鼠,末次加强免疫后取小鼠脾细胞与骨髓瘤细胞Sp2/0-Ag-14进行融合。通过HA和HI试验、间接ELISA试验反复筛选阳性克隆,进行3次以上亚克隆,共获得7株抗SIV H1N1亚型HA McAbs。把他们分别命名为8C4、8C6、9D6、8A4、8B1、9B2、11B4。运用分子生物学技术,从杂交瘤细胞株8C4中提取总RNA,经反转录、PCR分别分离了重链可变区(VH)基因,并进行了序列分析。获得7株抗SIV HA蛋白的单克隆抗体,其中8C4、8C6、9D6株具有血凝活性和中和活性。7株单抗腹水HI效价在512-256000之间,ELISA效价在16000-1024000。免疫球蛋白亚型试剂盒鉴定结果表明:8C4、8C6、9D6属于Ig2a亚型;8A4属于IgG1亚型;8B1属于IgG2b亚型。Western-blot分析结果显示:7株单抗能与SIV H1亚型的HA蛋白在72KD处反应。血凝试验表明8C4、8C6、9D6 3株单抗只与SIV H1N1和A/PR/8/34(H1N1)反应,而不与其他亚型的SIV、AIV、新城疫病毒(NDV)、鹅腺病毒等发生反应,表明这些McAbs能特异性识别SIV H1N1 HA蛋白而不与其他病毒反应。MDCK细胞中和试验表明:8C4、8C6、9D6 3株抗SIV H1亚型HA McAbs显示出较好的中和特性,也进一步说明了他们具有一定的中和能力和潜在的治疗功能。同样是具有HI活性的单抗却表现出不同的中和能力,这说明这些单抗之间存在着不同的抗原作用位点,这也为抗体的结构和功能性研究提供了条件。通过RT-PCR从单抗8C4中得到了VH基因。用BLAST和IMGT/V-QUEST数据库比对可知,8C4-VH1基因由375个核苷酸组成,编码125个氨基酸。包含4个FR区和CDR1、CDR2、CDR3三个高变区,CDR1含GYTFTSYY 8个氨基酸、CDR2含IYPGDGST 8个氨基酸、CDR3含ARVMIAMDY 9个氨基酸。第22位和96位半胱氨酸,形成链内特征性二硫键。重链可变区由VH、DH和JH 3个基因片段编码,DH基因编码重链V区大部分CDR3,JH基因片段连接V和C基因,编码包括重链V区CDR3除DH编码的其余部分和第4骨架区。由于可变区由几个基因片段编码,每个基因片段以基因群的方式存在,因此只有通过基因重排使各基因片段连接起来才能形成有功能的完整的可变区基因。获得的8C4的重链基因,V基因源于IgHV1S56*01,J基因源于IgHJ4*01,D基因可能有如下来源IgHD6-1*01或IgHD6-1*02或IGHD6-2*01或IgHD6-2*02或IgHD6-3*01,V、D、J 3个基因经重排后相互连接在一起,形成V-D-J复合体,完成重链重排。该序列符合鼠Ig重链基本框架结构,框架区内无终止密码子,是重排产生的序列。8C4-VH1同报告的序列号为IGHV1S56*01的同源率为96.88%,但IGHV1S56*01序列在抗体的高变区CDR3部分没有提交。同样采用RT-PCR方法从杂交瘤细胞8C4中获得的两个轻链基因8C4-VL1和8C4-VL2,经序列分析可知均是抗体轻链假基因。 HA蛋白特异性McAbs的研制为H1抗原变异分析及H1亚型猪流感诊断方法的建立奠定了物质基础。本实验获得的重链序列为研制单域抗体及进一步构建单链抗体奠定了物质基础。
[Abstract]:The HA gene was obtained from the virus by RT-PCR method, and the eukaryotic expression plasmid HA-pCI-neoc was constructed. The female BALB/c mice were immunized with eukaryotic expression plasmid HA-pCI-neo. The spleen cells of the mice were fused with Sp2/0-Ag-14 of myeloma cells after the last enhanced immunization. Through HA and HI tests, indirect ELISA test repeated screening of positive clones, more than 3 times of subcloning, a total of 7 strains resistant to SIV H1N1 subtype HA McAbs were obtained. They were named 8C4C6C6D6O8A4 8B1B2B2O11B4. respectively. Total RNAs were extracted from hybridoma cell line 8C4 by molecular biology technique. Heavy chain variable region (VH) genes were isolated and sequenced by reverse transcription-polymerase chain reaction (RT-PCR). Seven McAbs against SIV HA protein were obtained. Among them, 8C4C6C6D6 strain had hemagglutination activity and neutralizing activity. The HI titer of monoclonal antibody was 512-256000. The titer of Elisa was 16000-1024000. The results of immunoglobulin subtype assays showed that: 8C4C4C6C6D6 belongs to Ig2a subtype, 8A4 belongs to IgG1 subtype, 8B1 belongs to IgG2b subtype. Western-blot analysis showed that the McAb of the 7 strains could react with the HA protein of SIV H1 subtype in 72KD. The results of hemagglutination test showed that the McAb of 8C4C6 / 9D63 only reacted with SIV H1N1 and APR-8 / 34 H1 / N1, but did not react with other subtypes of SIVAIV, NDV, adenovirus, etc. The results showed that these McAbs could specifically recognize the HA protein of SIV H1N1 without reacting with other viruses. The neutralization test showed that the SIV H1 subtype HA McAbs showed a good neutralization property of the strain: 8C4C4C6C6C9D63. It also shows that they have certain neutralization ability and potential therapeutic function. The same monoclonal antibody with HI activity showed different neutralization ability, which indicated that there were different antigenic sites among these McAbs, which provided conditions for the study of the structure and function of antibodies. VH gene was obtained from monoclonal antibody 8C4 by RT-PCR. The results of BLAST and IMGT/V-QUEST database showed that the 8C4-VH1 gene was composed of 375 nucleotides and encoded 125 amino acids. CDR1 contains GYTFTSYY 8 amino acids and CDR2 contains IYPGDGST 8 amino acids and CDR3 contains 9 ARVMIAMDY amino acids. Cysteine at the 22 th and 96 th position forms the characteristic disulfide bond in the chain. The variable region of heavy chain is encoded by three fragments of VHH DH and JH genes. Most of the heavy chain V region of CDR3N JH gene fragment is linked to V and C genes. The coding includes the other parts of heavy chain V region CDR3 except DH coding and the 4th skeleton region. Because the variable region is encoded by several gene fragments, each gene fragment exists in the form of gene group. Therefore, only by rearranging each gene fragment together can a functional complete variable region gene be formed. The obtained 8C4 heavy chain gene V gene was derived from the IgHV1S5601FG gene derived from the IgHJ4F01D gene, which may have the following three genes: IgHD6-1*01 or IgHD6-1*02 or IGHD6-2*01 or IgHD6-2*02 or IgHD6-3C01VDNJ gene rearranged together to form V-D-J complex and complete the rearrangement of the heavy chain. The sequence is in accordance with the basic frame structure of mouse Ig heavy chain, and there is no stop codon in the frame region. The sequence. 8C4-VH1 is the rearranged sequence. The homology of the sequence with the reported sequence number IGHV1S56*01 is 96.88, but the IGHV1S56*01 sequence is not submitted in the CDR3 part of the highly variable region of the antibody. Two light chain genes 8C4-VL1 and 8C4-VL2 obtained from 8C4 of hybridoma cells by RT-PCR method were identified as light chain pseudogenes by sequence analysis. The preparation of HA protein-specific McAbs laid a solid foundation for the analysis of H1 antigen variation and the establishment of diagnostic method for H1 subtype swine flu. The heavy chain sequence obtained in this experiment laid a solid foundation for the development of single domain antibody and the further construction of single chain antibody.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

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