杂交瘤细胞株功能性与非功能性V_K基因结构分析

Sequence analysis of functional and nonfunctional V K genes from a hybridoma

目的：探讨不同引物从杂交瘤细胞株中扩增出功能性和非功能性ＶＫ基因的关系。方法：利用ＲＴＰＣＲ技术扩增出轻链可变区基因，测序后，利用ＤＮＡＳＩＳ７和与ＧｅｎＢａｎｋ比较分析。结果：发现引物（ＶＬ５′．１和ＪＫ１／２）扩增出了非功能性ＶＫ基因。而改用引物（ＶＬ５′．２和ＶＬ３′）后，扩增出了有功能的ＶＫ基因。结论：非功能性ＶＫ基因的扩增，说明部分杂交瘤细胞出现了来源于原融合细胞的基因异常重排。在制备基因工程抗体中，区别功能性和非功能性ＶＫ基因具有重要意义。

Objective:To find the relationship between functional and non functional V K genes which were cloned from a hybridoma(7579) with two different kinds of family specific primers.Methods:From a secreting mouse hybridoma cell line 7579, total RNA was prepared and was reverse transcribed into the first strand of cDNAs. The V L genes was amplified by PCR with family specific primers, and then cloned into the pGEM T vectors in the competent cell JM109. With the dye terminator cycle sequencing method, the V L gene was sequenced. The results was analyzed with DNASIS7, and also was compared with the NIH's GenBank .Results:A non functional V K, which was amplified with the primers V L5′.1 and J K1/2, was found. It exhibited 98% homology to the BALB/c V K 21 E. However there are 4 bp deletion at it's V/J junction leading to a frame shift. This results in a premature termination codon at following position. Using different family specific primers, a functional V K gene was obtained. Conclusion:Analysis of the two kinds of V K genes suggests that the functional V K gene is derived from the B cell and the non functional arises from an aberrent mRNA transcript which is present in all standard fusion partners derived from the original MOPC 21 tumor.