抗CD45单克隆抗体可变区基因的克隆及序列分析
发布时间：2019-05-12 10:15
【摘要】： 目的抗CD45单克隆抗体可变区基因的克隆及序列分析,为进一步的基因工程抗体改造研究奠定基础。 方法 1.杂交瘤细胞总RNA的提取:以异硫氰酸胍-苯酚-氯仿法提取总RNA。 2.引物的设计:利用V区N端相对保守的特点,根据Ig可变区的序列,在N端和恒定区CH1合成5’和3’端兼并的“通用”引物。 3. McAbV区基因的扩增:以RNA为模板,采用cDNA合成试剂盒合成第一条链cDNA,以cDNA为模板,用TaqDNA酶扩增McAbV区基因。 4. V区基因核苷酸序列分析:用胶回收试剂盒回收PCR产物后,将McAb的VH基因和VL基因分别与pGEM-T载体连接,并转化大肠杆菌,经菌落PCR鉴定阳性菌。每一种连接产物挑取3个阳性菌进行序列分析。 5.利用DNAtools,IMGT/QUEST及EBI TOOLS:ClustalW2分析软件对轻链和重链基因分别进行同源性比较。 结果 1.第一次PCR 用2条重链上游引物(MH1,MH2),一条下游引物(IgG1-C 3’);轻链上(MK)下(Kc)游各一条引物进行扩增,测序结果获得编码单克隆抗体可变区的核苷酸序列。含引物的VH长397bp,VL长377bp,此次扩增出的基因序列无信号肽序列,适于ScFv的构建,进一步以更换引物进行二次PCR,来扩增含信号肽序列的基因。 2.第二次PCR 用3条重链上游(VH5’1,2,3),一条下游引物(IgG1-C 3’);3条轻链上游(VL5’1,2,3)引物,下游引物(Kc)进行扩增,测序结果:VH为有功能的基因片段,长348bp,编码116个氨基酸,在VH前有57bp的信号肽序列,编码19个氨基酸;含引物的Vκ长369bp,扩增的为非功能性Vκ链。经分析发现扩增出非功能轻链的上游引物为VL5’1,为了扩增出有功能的轻链基因,需重新设计引物。 3.第三次PCR 用新合成的VL5’4,5取代原轻链上游引物VL5’1,用4条轻链上游引物(VL5’2,3,4,5),新合成下游引物(VL3’1),扩增出的Vκ核苷酸序列长333bp,编码111个氨基酸,在Vκ前有57bp的信号肽序列,编码19个氨基酸,经IMGT/QUEST系统分析为有功能的轻链基因。 对所扩增的Ig基因进行同源性比较结果:抗CD45单抗有功能的轻链均属于IGκV1-117’01家族;功能性重链属于IGHV2-9-1’01家族。其中扩增出的非功能的轻链属于IGκV3-12’01家族。 结论成功获取抗CD45单抗的轻链与重链基因,以及含有信号肽的基因,为进一步通过基因工程技术改造抗体奠定了良好的基础。
[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，
本文编号：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，
本文编号：2475307
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