本文選題：單克隆抗體 + H1N1豬流感��； 參考：《東北農(nóng)業(yè)大學(xué)》2009年碩士論文

【摘要】： 用RT-PCR方法從病毒中獲得HA基因,構(gòu)建真核表達(dá)質(zhì)粒HA-pCI-neo。以真核表達(dá)質(zhì)粒HA-pCI-neo肌肉免疫6-8w雌性BALB/c小鼠,末次加強免疫后取小鼠脾細(xì)胞與骨髓瘤細(xì)胞Sp2/0-Ag-14進(jìn)行融合。通過HA和HI試驗、間接ELISA試驗反復(fù)篩選陽性克隆,進(jìn)行3次以上亞克隆,共獲得7株抗SIV H1N1亞型HA McAbs。把他們分別命名為8C4、8C6、9D6、8A4、8B1、9B2、11B4。運用分子生物學(xué)技術(shù),從雜交瘤細(xì)胞株8C4中提取總RNA,經(jīng)反轉(zhuǎn)錄、PCR分別分離了重鏈可變區(qū)(VH)基因,并進(jìn)行了序列分析。 獲得7株抗SIV HA蛋白的單克隆抗體,其中8C4、8C6、9D6株具有血凝活性和中和活性。7株單抗腹水HI效價在512-256000之間,ELISA效價在16000-1024000。免疫球蛋白亞型試劑盒鑒定結(jié)果表明:8C4、8C6、9D6屬于Ig2a亞型;8A4屬于IgG1亞型;8B1屬于IgG2b亞型。Western-blot分析結(jié)果顯示:7株單抗能與SIV H1亞型的HA蛋白在72KD處反應(yīng)。血凝試驗表明8C4、8C6、9D6 3株單抗只與SIV H1N1和A/PR/8/34(H1N1)反應(yīng),而不與其他亞型的SIV、AIV、新城疫病毒(NDV)、鵝腺病毒等發(fā)生反應(yīng),表明這些McAbs能特異性識別SIV H1N1 HA蛋白而不與其他病毒反應(yīng)。MDCK細(xì)胞中和試驗表明:8C4、8C6、9D6 3株抗SIV H1亞型HA McAbs顯示出較好的中和特性,也進(jìn)一步說明了他們具有一定的中和能力和潛在的治療功能。同樣是具有HI活性的單抗卻表現(xiàn)出不同的中和能力,這說明這些單抗之間存在著不同的抗原作用位點,這也為抗體的結(jié)構(gòu)和功能性研究提供了條件。 通過RT-PCR從單抗8C4中得到了VH基因。用BLAST和IMGT/V-QUEST數(shù)據(jù)庫比對可知,8C4-VH1基因由375個核苷酸組成,編碼125個氨基酸。包含4個FR區(qū)和CDR1、CDR2、CDR3三個高變區(qū),CDR1含GYTFTSYY 8個氨基酸、CDR2含IYPGDGST 8個氨基酸、CDR3含ARVMIAMDY 9個氨基酸。第22位和96位半胱氨酸,形成鏈內(nèi)特征性二硫鍵。重鏈可變區(qū)由VH、DH和JH 3個基因片段編碼,DH基因編碼重鏈V區(qū)大部分CDR3,JH基因片段連接V和C基因,編碼包括重鏈V區(qū)CDR3除DH編碼的其余部分和第4骨架區(qū)。由于可變區(qū)由幾個基因片段編碼,每個基因片段以基因群的方式存在,因此只有通過基因重排使各基因片段連接起來才能形成有功能的完整的可變區(qū)基因。獲得的8C4的重鏈基因,V基因源于IgHV1S56*01,J基因源于IgHJ4*01,D基因可能有如下來源IgHD6-1*01或IgHD6-1*02或IGHD6-2*01或IgHD6-2*02或IgHD6-3*01,V、D、J 3個基因經(jīng)重排后相互連接在一起,形成V-D-J復(fù)合體,完成重鏈重排。該序列符合鼠Ig重鏈基本框架結(jié)構(gòu),框架區(qū)內(nèi)無終止密碼子,是重排產(chǎn)生的序列。8C4-VH1同報告的序列號為IGHV1S56*01的同源率為96.88%,但I(xiàn)GHV1S56*01序列在抗體的高變區(qū)CDR3部分沒有提交。同樣采用RT-PCR方法從雜交瘤細(xì)胞8C4中獲得的兩個輕鏈基因8C4-VL1和8C4-VL2,經(jīng)序列分析可知均是抗體輕鏈假基因。 HA蛋白特異性McAbs的研制為H1抗原變異分析及H1亞型豬流感診斷方法的建立奠定了物質(zhì)基礎(chǔ)。本實驗獲得的重鏈序列為研制單域抗體及進(jìn)一步構(gòu)建單鏈抗體奠定了物質(zhì)基礎(chǔ)。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392.1

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