摘要
用脂多糖诱导正常人外周血单核细胞使产生粒细胞集落刺激因子。从诱导细胞中提取出总RNA,以特异引物经逆转录PCR扩增出粒细胞集落刺激因子mRNA的cDNA,将其插入经改造的分泌型表达载体pIN-omp A,并转化受体菌E.coli。克隆的cDNA通过限制性酶双酶切和3′端、5′端及中间序列探针分子杂交鉴定,结果与预期一致。表达质粒在大肠杆菌中经IPTG诱导,粒细胞集落刺激因子被分泌到细菌的周质中。产物提取后,用双单抗夹心ELISA法检测具有粒细胞集落刺激因子的免疫活性。
Monocytes were isolated from freshly prepared peripheral blood of a healthy adult human and were stimulated by lipopolysaccharide (LPS) to produce hG-CSF. The hG-CSF appeared in culture after 12 hours induction and to be maximum through 24 hours. The total RNA were isolated from human monocytes induced by LPS by using the method of acid guaninium-phenol-chloroform extraction and amplified specifically by reverse transcription (RT-PCR) technique to obtain hG-CSF cDNA. The optimal annealing temperature determined experimentally was 57℃, at which the reaction product for hG-GSF cDNA was maximized and non-specific products were reduced. The hG-CSF cDNA was inserted into the secretion expression rector pIN-ompA reformed by the laboratory, and then transformed competent E. coli. Recombinant plasmids were identified by restriction enzyme assay and Southern hybridization with 3' terminal, 5' terminal and middle sequence probes. After induction using IPTG, the hG-CSF protein was detected in the periplasmic space of the recombinant E. coli cells. These data provide evidence that hG-CSF cDNA has been cloned and expressed in E. coli.
出处
《北京大学学报（自然科学版）》
CAS
CSCD
北大核心
1993年第6期675-679,共5页
Acta Scientiarum Naturalium Universitatis Pekinensis
关键词
CDNA
hG-CSF
无性系
hG-CSF
cDNA cloning
Secretion expression vector
