摘要
用异硫氰酸胍法从分泌单克隆抗体的杂交瘤细胞中提取总RNA,经oligo(dT)-纤维素柱亲和层析获得poly(A)^+ RNA后,用恒定区5′端第122—125号氨基酸密码的互补序列3′A-T-A-G-G-T-G-A-C-C 5′做为引物,进行逆转录酶反应,合成双链cDNA,大小为300bp左右,与重链可变区基因的长度相符。用dC:dG接尾的方法,将ds-cDNA插入pUC19质粒,转化E.coli HB101。分离出重组体之后,经菌落原位杂交,酶切重组质粒DNA及Southern印迹,证明插入片段是重链可变区基因。
With the guanidinium isothiocyanate method,total RNA was isolated from hybridoma cells that secrete monoclonal antibody against Brucella melitenses.Poly (A)+RNA was obtained by oligo (dT)-cellulose affinity chromatography.Reverse transcriptase reaction was performed with a primer 3'A-T-A-G-G-T-G-A-C-C 5' that is complement to the codons of No.122-125 amino acid residues in 5' terminus of constant region.The size of synthesized ds-cDNA is about SOObp,that is consistent with the length of variable region genes of heavy chain.The ds-cDNA was inserted into plasmid pUC19 with dC:dG tailing method,and the inserted plasmid was used to transform E.colt HB101.It has been proved that the insert was a variable region gene of heavy chain by clone hybridization in situ,size of insert and Southern blot.
