论文部分内容阅读
获得抗体的可变区基因是由鼠源性ＭｃＡｂ制备重组抗体的前提，本实验合成与轻链可变区（ＶＬ）ＦＲ１和ＦＲ４互补的通用引物，由分泌抗人Ｃ１－ＩＮＨＭｃＡｂ的杂交瘤Ｆ７细胞株提取总ＲＮＡ，经ＲＴ－ＰＣＲ扩增出Ｆ７ＶＬ的ｃＤＮＡ片段，并将其克隆入ｐＵＣ１８／１９测序载体，从两端进行双脱氧核苷酸随机终止法的ＤＮＡ序列测定。结果显示：ＶＬｃＤＮＡ是由３０３个碱基组成，编码１０１个氨基酸残基。由国际联机检索进行ＥＭＢＬ和Ｋａｂａｔ基因库扫描发现：Ｆ７ＶＬ仅与Ｉｇ同源，符合小鼠Ｉｇ的ＶＬ基因特征，同源性为６０％～８０％。根据Ｋａｂａｔ分类方法，Ｆ７ＶＬ基因推导的氨基酸序列应归属于小鼠Ｉｇ的ＶＬ基因ＩＶ亚组，是由Ｖｋ－Ｊｋ１重排产生。ＣＤＲ３含有９个氨基酸残基，其中含３个不相同氨基酸残基，ＶＬ大多数的氨基酸变化集中在ＦＲ１和ＣＤＲ１区，Ｆ７ＶＬ符合ＩｇＶＬ氨基酸残基变化的基本特征。Ｃｙｓ２３和Ｃｙｓ８８残基位于Ｆ７ＶＬＣＤＲ１和ＣＤＲ３的起始点，与二硫键形成有关。成功获得Ｆ７ＶＬ基因为进一步构建和表达单链Ｆｖ（ｓｃＦｖ）抗体打下了良好的基础。
The variable region gene of the obtained antibody is a premise for preparing the recombinant antibody from murine McAb. In this experiment, universal primer complementary to the variable region of the light chain (VL) FR1 and FR4 was synthesized, which was composed of the hybridoma F7 secreting anti-human C1-INHMcAb The total RNA was extracted from the cell lines. The cDNA fragment of F7VL was amplified by RT-PCR and cloned into the pUC18 / 19 sequencing vector. The DNA sequencing of the dideoxynucleotide termination assay was carried out from both ends. The results showed that VLcDNA consisted of 303 bases and encoded 101 amino acid residues. The international online search for EMBL and Kabat gene bank found: F7VL only homologous with Ig, in line with the characteristics of mouse Ig VL gene, homology of 60% to 80%. According to the Kabat classification method, the deduced amino acid sequence of the F7VL gene belongs to the IV subgroup of the VL gene of mouse Ig and is produced by the rearrangement of Vk-Jk1. CDR3 contains 9 amino acid residues, which contain 3 different amino acid residues. The majority of VL amino acid changes are concentrated in the FR1 and CDR1 regions. The F7VL conforms to the basic characteristics of the amino acid residues of IgVL. The Cys23 and Cys88 residues are located at the start of F7VLCDR1 and CDR3 and are involved in disulfide bond formation. The successful F7VL gene has laid a good foundation for further construction and expression of single-chain Fv (scFv) antibody.