摘要
克隆人 IL - 1 2 ( h IL - 1 2 ) p40和 p35亚单位 c DNA,构建人单链 IL- 1 2 ( rhsc IL- 1 2 )融合基因 ,并在哺乳动物细胞中进行表达。方法 从经 PDBu刺激的 EBV转化的人 B淋巴母细胞株 NC37中提取 m RNA,经 RT- PCR分别获得 h IL- 1 2 p40和 p35亚单位编码序列的 c DNA,运用重组 PCR技术将两段基因通过一疏水性多肽接头 ( Gly4 Ser) 3 DNA序列进行体外基因重组 ,构建 rhsc IL- 1 2融合基因 ,将其插入 pc DNA3.1 ( + )真核表达载体 ,经脂质体转染 COS7细胞进行表达 ,Western blot进行分析。结果 所克隆的 h IL- 1 2 p40、p35c DNA序列和构建的 rhsc IL - 1 2融合基因 DNA序列均经测序得以证实 ,融合基因可在 COS7中表达其产物 rhsc IL- 1 2融合蛋白 ,其分子量为 70 KD,可与鼠抗人 IL - 1 2 p40 /p70单克隆抗体特异性结合。结论 本研究结果为进一步探讨 rhsc IL- 1 2融合蛋白的生物学活性和特性奠定了基础 ,也为 rhsc IL- 1
Objective Cloning of p40 and p35 subunit cDNA of human IL 12(hIL 12), constructing of fusion gene of recombinant human single chain IL 12(rhscIL 12) and expression of the fusion gene in mammalian cell.Methods Extracting mRNA from human lymphoblastoid cell line NC37 stimulated by PDBu for cloning hIL 12 p40 and p35 cDNA by RT PCR respectively ,applying a hydrophobic polypeptide linker(Gly 4Ser) 3 for splicing two different gene fragments (hIL 12 p40 linker p35)by recombinant PCR for constructing rhscIL 12 fusion gene in vitro ,inserting rhscIL 12 fusion gene into pcDNA3.1(+) eukarytic expressing plasmid,transfecting pcDNA3.1(+) rhscIL 12 into COS7 cell by lipofection for expressing the fusion protein, analysing with Western blot.Results The sequences of cloned hIL 12 p40,p35 cDNA and constructed rhscIL 12 fusion gene were verified by sequencing,mRNA and fusion protein of rhscIL 12 fusion gene were expressed characteristically in COS7 cell.Western blot showed the appearance of 70KD band.The fuctional fusion protein combined with mouse anti human IL 12 monoclonal antibody specifically.Conclusion This research has paved the way for further study on biological functions of rhscIL 12 fusion gene, and also offers a possibility of expressing rhscIL 12 fusion protein in prokaryotic cells.
出处
《西安医科大学学报》
CSCD
2000年第4期297-302,共6页
Journal of Xi'an Medical University(Chinese)
基金
陕西省自然科学基金! ( 99SM4 9)
关键词
重组人单链白细胞介素12
融合基因
真核表达
human interleukin 12(hIL 12)
recombinant human single chain interleukin 12
