细胞内RT－PCR扩增免疫球蛋白重链可变区基因  

In-Cell Reverse Transcription PolymeraseChain Reaction(In-cell-RT-PCR):Amplify-ing the Reverse Transcripted lmmunoglobulinHeavy Chain Gene Fragment within Hybrido-ma Cells.

作　　者：李昌龙[1] 赵满仓[1] 王抒[1] 蒋雷[1] 李健斋[1] 

机构地区：[1]卫生部北京老年医学研究所,解放军石家庄空军医院

出　　处：《生物化学与生物物理进展》1995年第4期380-382,共3页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金

摘　　要：通常，逆转录ＰＣＲ（ＲＴ－ＰＣＲ）需要高质量的ｍＲＮＡ，操作过程复杂，效率低且易受到ＲＮａｓｅ的破坏，为了简化操作，提高效率，用１０％甲醛盐溶液固定杂交瘤细胞，以Ｎｐ－４０渗透化处理细胞后做ＲＴ－ＰＣＲ，获得了大约３５０ｂｐ的特异性免疫球蛋白重链可变区基因片段，与用同一对引物得到的常规ＲＴ－ＰＣＲ扩增产物一致．这项技术可用于获得特定结构基因片段，连结并扩增嵌合蛋白基因及构建多克隆免疫球蛋白文库等．General reverse transcription poly-merase chain reaction(RT-PCR)is a compli-cated process with low efficiency.And highquality mRNA is required and the mRNA iseasily destroyed by RNase.The hybridomacells were fixed with 10%formaldehyde solu-tion in normal saline and permeabilised by-0.5%Nonidet P-40 in water. After themRNA was reversely transcribed to cDNAand the cDNA was amplified by polymerasechain reaction(PCR),a specific heavy chainvariable region gene(VH)of immunoglobulinwith length of about 350 bp,being consistentwith the product amplified by normal RT-PCR,was obtained.This technique could beapplied to prepare specific structure gene frag-ments,to link and amplify chimeric proteingenes and to construct human antibody li-braries.

关 键 词：细胞 RT-PCR 重链可变区基因 免疫球蛋白 

分 类 号：Q26[生物学—细胞生物学]