粒细胞-巨噬细胞集落刺激因子与HBsAg融合蛋白基因的分子克隆及序列测定

Cloning and sequencing of fusion protein gene of recombinant GM-HBsAg

目的研究粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）的免疫活性。方法用聚合酶链反应（ＰＣＲ）技术扩增出粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）３８４ｂｐ的基因片段及ＨＢｓＡｇ８４６ｂｐ基因片段，扩增产物经柱纯化后，由Ｔ４ＤＮＡ酶连结为１２３０ｂｐ的片段，将此片段命名为ＬＧＨ，克隆到ｐＵＣ１９质粒中，利用菌落ＰＣＲ法快速筛选阳性克隆及限制酶切鉴定插入片段的大小。结果该基因全长１２３０ｂｐ，经序列分析，证实为ＧＭ－ＣＳＦ－ＨＢｓＡｇ基因。结论ＧＭ－ＨＢｓＡｇ融合蛋白基因构建成功，为进一步研究其生物学活性及表达奠定了基础。

GM-CSF is an effective adjuvant for immunology, it was used to improve the immunological function of antigen and to increase the anti-HBs responses. Through using polymerase chain reaction (PCR) technique, we amplified 384bp and 846bp gene fragments from GM-CSF and HBsAg. The products were extracted and purified; a 1230bp gene fragment was obtained by T4 DNA linkage. This amplified product was cloned into pUC19 vector. The positive clones were scereened and the inserted fragment was determined by restriction enzymes. The gene fragment length determined is 1230bp, the sequence analysis showed that the gene fragment is GM-CSF-HBsAg gene. The successful construction of GM-HBsAg fusion protein gene provides basis for research of its biologieal activity and expression.