人外周血IL-12 P35 cDNA的巢式PCR扩增、克隆与测序

目的 克隆中国汉族人IL—12P35 cDNA序列，并进行序列测定。方法 利用反转录巢式PCR从健康人外周血总RNA中扩增IL—12P35 cDNA序列，插入T载体PMD—T中，重组质粒转化大肠杆菌JM109，所获克隆经PCR初筛后进行酶切鉴定，阳性克隆进行DNA测序。结果 从健康人外周血总DNA中扩增出约670bp条带，与预期大小相同，PCR与酶切鉴定证明得到PMD／P35阳性克隆，DNA测序证实了插入片段的正确性。结论 在体外成功的扩增、克隆了中国汉族人IL—12P35 cDNA序列。为继续开展IL—12的基础与应用研究奠定了基础。

Nested PCR amplification,cloning and sequence determination of the cDNA of human IL-12 P35

Objective Cloning and sequencing the cDNA of IL-2 P35 of Chinese Han Nationality. Methods The IL-2 P35 cDNA was amplified by RT-nested PCR from the total RNA of peripheral blood of healthy Chinese Han Nationality.After purification,the gene fragment was inserted into the PMD-T vector.The recombinant plasmid was transformed into E.Coli JM109.Positive clones were screened and identified by PCR first and subsequently confirmed by endonuclease digestion.The sequencing of the inserted fragment was also determined. Results About 670bp DNA fragment was amplified and the total RNA of peripheral blood of healthy people as expected.The positive recombinant was identified by PCR and endonuclease digestion.The sequence of insert fragment also indicated the right recombinant achieved. Conclusion The IL-12 P35 cDNA was successfully amplified and cloned.That would promote the subsequent elementary and applied research on IL-12.