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 polymerase chain reaction(RT-PCR)is a complicated process with low efficiency.And high quality mRNA is required and the mRNA is easily destroyed by RNase.The hybridoma cells were fixed with 10% formaldehyde solution in normal saline and permeabilised by 0.5% Nonidet P-40 in water. After the mRNA was reversely transcribed to cDNA and the cDNA was amplified by polymerase chain reaction(PCR),a specific heavy chain variable region gene(VH)of immunoglobulin with length of about 350 bp,being consistent with the product amplified by normal RT-PCR,was obtained. This technique could be applied to prepare specific structure gene fragmenu,to link and amplify chimeric protein genes and to construct human antibody lihraries.
李昌龙,赵满仓,王抒,蒋雷,李健斋.细胞内RT-PCR扩增免疫球蛋白重链可变区基因[J].生物化学与生物物理进展,1995,22(4):380-382
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