抗CD45單克隆抗體可變區(qū)基因的克隆及序列分析
發(fā)布時間：2019-05-12 10:15
【摘要】： 目的抗CD45單克隆抗體可變區(qū)基因的克隆及序列分析,為進一步的基因工程抗體改造研究奠定基礎(chǔ)。 方法 1.雜交瘤細胞總RNA的提取:以異硫氰酸胍-苯酚-氯仿法提取總RNA。 2.引物的設(shè)計:利用V區(qū)N端相對保守的特點,根據(jù)Ig可變區(qū)的序列,在N端和恒定區(qū)CH1合成5’和3’端兼并的“通用”引物。 3. McAbV區(qū)基因的擴增:以RNA為模板,采用cDNA合成試劑盒合成第一條鏈cDNA,以cDNA為模板,用TaqDNA酶擴增McAbV區(qū)基因。 4. V區(qū)基因核苷酸序列分析:用膠回收試劑盒回收PCR產(chǎn)物后,將McAb的VH基因和VL基因分別與pGEM-T載體連接,并轉(zhuǎn)化大腸桿菌,經(jīng)菌落PCR鑒定陽性菌。每一種連接產(chǎn)物挑取3個陽性菌進行序列分析。 5.利用DNAtools,IMGT/QUEST及EBI TOOLS:ClustalW2分析軟件對輕鏈和重鏈基因分別進行同源性比較。 結(jié)果 1.第一次PCR 用2條重鏈上游引物(MH1,MH2),一條下游引物(IgG1-C 3’);輕鏈上(MK)下(Kc)游各一條引物進行擴增,測序結(jié)果獲得編碼單克隆抗體可變區(qū)的核苷酸序列。含引物的VH長397bp,VL長377bp,此次擴增出的基因序列無信號肽序列,適于ScFv的構(gòu)建,進一步以更換引物進行二次PCR,來擴增含信號肽序列的基因。 2.第二次PCR 用3條重鏈上游(VH5’1,2,3),一條下游引物(IgG1-C 3’);3條輕鏈上游(VL5’1,2,3)引物,下游引物(Kc)進行擴增,測序結(jié)果:VH為有功能的基因片段,長348bp,編碼116個氨基酸,在VH前有57bp的信號肽序列,編碼19個氨基酸;含引物的Vκ長369bp,擴增的為非功能性Vκ鏈。經(jīng)分析發(fā)現(xiàn)擴增出非功能輕鏈的上游引物為VL5’1,為了擴增出有功能的輕鏈基因,需重新設(shè)計引物。 3.第三次PCR 用新合成的VL5’4,5取代原輕鏈上游引物VL5’1,用4條輕鏈上游引物(VL5’2,3,4,5),新合成下游引物(VL3’1),擴增出的Vκ核苷酸序列長333bp,編碼111個氨基酸,在Vκ前有57bp的信號肽序列,編碼19個氨基酸,經(jīng)IMGT/QUEST系統(tǒng)分析為有功能的輕鏈基因。 對所擴增的Ig基因進行同源性比較結(jié)果:抗CD45單抗有功能的輕鏈均屬于IGκV1-117’01家族;功能性重鏈屬于IGHV2-9-1’01家族。其中擴增出的非功能的輕鏈屬于IGκV3-12’01家族。 結(jié)論成功獲取抗CD45單抗的輕鏈與重鏈基因,以及含有信號肽的基因,為進一步通過基因工程技術(shù)改造抗體奠定了良好的基礎(chǔ)。
[Abstract]:Objective to clone and sequence analysis of variable region gene of monoclonal antibody against CD45, so as to lay a foundation for further research on modification of genetic engineering antibody. Method 1. Extraction of Total RNA from hybridoma cells by guanidine isothiocyanate-Phenol-chloroform method 2. Design of primers: according to the sequence of Ig variable region, the "universal" primers merged at 5 'and 3' ends were synthesized by CH1 in N-terminal and constant region, taking advantage of the relatively conservative characteristics of N-terminal in V region. 3. Amplification of McAbV region gene: using RNA as template, the first chain cDNA, was synthesized by cDNA synthesis kit and cDNA was used as template, and McAbV region gene was amplified by TaqDNA enzyme. 4. Nucleotides sequence analysis of V region gene: after PCR products were recovered by gel recovery kit, the VH gene and VL gene of McAb were ligated with pGEM-T vector and transformed into E. coli. The positive bacteria were identified by colony PCR. Three positive bacteria were selected from each junction product for sequence analysis. 5. The homology of light chain gene and heavy chain gene was compared by DNAtools,IMGT/QUEST and EBI TOOLS:ClustalW2 software. Result 1. For the first time, two heavy chain upstream primers (MH1,MH2) and one downstream primer (IgG1-C 3') were used for the first PCR. One primer of (Kc) under light chain (MK) was amplified, and the nucleotides encoding the variable region of monoclonal antibody were obtained by sequencing. The VH with primers was 397bp and VL was 377bp. the amplified gene sequence was non-signal peptide sequence and was suitable for the construction of ScFv. The gene containing signal peptide sequence was amplified by secondary PCR, with primers. 2. For the second PCR, three heavy chains upstream (VH5'1,2,3) and one downstream primer (IgG1-C 3') were used. Three light chain upstream (VL5'1,2,3) primers and downstream primers were amplified by (Kc). The results of sequencing showed that VH was a functional gene fragment with a length of 348bp. it encoded 116 amino acids. There was a signal peptide sequence of 57bp before VH, encoding 19 amino acids. The length of V K with primers was 369bp, and the amplified V K chain was nonfunctional V K chain. It is found that the upstream primers for amplification of non-functional light chains are VL5'1,. In order to amplify functional light chain genes, primers need to be redesigned. 3. In the third PCR, the newly synthesized VL5'4,5 was used to replace the original light chain upstream primer VL5'1, with four light chain upstream primers (VL5'2,3,4,5) and the newly synthesized downstream primers (VL3'1). The amplified V K nucleotides were 333bp. It encodes 111amino acids, has 57bp signal peptide sequence before V K, and encodes 19 amino acids. It is a functional light chain gene analyzed by IMGT/QUEST system. The homology of the amplified Ig gene was compared. the results showed that the functional light chain of anti-CD45 monoclonal antibody belonged to IGkV 1-117 鈮，
本文編號：2475307
[Abstract]:Objective to clone and sequence analysis of variable region gene of monoclonal antibody against CD45, so as to lay a foundation for further research on modification of genetic engineering antibody. Method 1. Extraction of Total RNA from hybridoma cells by guanidine isothiocyanate-Phenol-chloroform method 2. Design of primers: according to the sequence of Ig variable region, the "universal" primers merged at 5 'and 3' ends were synthesized by CH1 in N-terminal and constant region, taking advantage of the relatively conservative characteristics of N-terminal in V region. 3. Amplification of McAbV region gene: using RNA as template, the first chain cDNA, was synthesized by cDNA synthesis kit and cDNA was used as template, and McAbV region gene was amplified by TaqDNA enzyme. 4. Nucleotides sequence analysis of V region gene: after PCR products were recovered by gel recovery kit, the VH gene and VL gene of McAb were ligated with pGEM-T vector and transformed into E. coli. The positive bacteria were identified by colony PCR. Three positive bacteria were selected from each junction product for sequence analysis. 5. The homology of light chain gene and heavy chain gene was compared by DNAtools,IMGT/QUEST and EBI TOOLS:ClustalW2 software. Result 1. For the first time, two heavy chain upstream primers (MH1,MH2) and one downstream primer (IgG1-C 3') were used for the first PCR. One primer of (Kc) under light chain (MK) was amplified, and the nucleotides encoding the variable region of monoclonal antibody were obtained by sequencing. The VH with primers was 397bp and VL was 377bp. the amplified gene sequence was non-signal peptide sequence and was suitable for the construction of ScFv. The gene containing signal peptide sequence was amplified by secondary PCR, with primers. 2. For the second PCR, three heavy chains upstream (VH5'1,2,3) and one downstream primer (IgG1-C 3') were used. Three light chain upstream (VL5'1,2,3) primers and downstream primers were amplified by (Kc). The results of sequencing showed that VH was a functional gene fragment with a length of 348bp. it encoded 116 amino acids. There was a signal peptide sequence of 57bp before VH, encoding 19 amino acids. The length of V K with primers was 369bp, and the amplified V K chain was nonfunctional V K chain. It is found that the upstream primers for amplification of non-functional light chains are VL5'1,. In order to amplify functional light chain genes, primers need to be redesigned. 3. In the third PCR, the newly synthesized VL5'4,5 was used to replace the original light chain upstream primer VL5'1, with four light chain upstream primers (VL5'2,3,4,5) and the newly synthesized downstream primers (VL3'1). The amplified V K nucleotides were 333bp. It encodes 111amino acids, has 57bp signal peptide sequence before V K, and encodes 19 amino acids. It is a functional light chain gene analyzed by IMGT/QUEST system. The homology of the amplified Ig gene was compared. the results showed that the functional light chain of anti-CD45 monoclonal antibody belonged to IGkV 1-117 鈮，
本文編號：2475307
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