CAJ | 학술논문

我们按照hBDNF基因全长编码序列设计合成引物，从人基因组DNA中扩增出76 0bp的片段，反向插入到pGEM-3Zf（+）载体上，获得pGEMBF18克隆，限制性酶分析和DNA序列测定均证实该克隆插入片段为hBDNF基因全长编码序列。从pGEMBF18克隆中获取hBDNF全长编码片段，与原核表达载体pGEX-5T连接，构建了p5TBF34原核表达重组质粒。重组质粒转化大肠杆菌JM109，经IPTG诱导表达，SDS-PAGE特异区带分子量为43kDa，此重组蛋白占菌体可溶性蛋白总量的7.53%, Western杂交证实该特异区带具hBDNF抗原活性。
아문안조hBDNF기인전장편마서렬설계합성인물，종인기인조DNA중확증 출76 0bp적편단，반향삽입도pGEM-3Zf（+）재체상，획득pGEMBF18극륭，한제성매분석화DNA서 렬측정균증실해극륭삽입편단위hBDNF기인전장편마서렬。종pGEMBF18극륭중획취hBDNF전장 편마편단，여원핵표체재체pGEX-5T련접，구건료p5TBF34원핵표체중조질립。중조질립전 화대장간균JM109，경IPTG유도표체，SDS-PAGE특이구대분자량위43kDa，차중조단백점균 체가용성단백총량적7.53%, Western잡교증실해특이구대구hBDNF항원활성。
The primers specific for the full-length BDNF coding sequence was designed and synthesized. The BDNF coding sequence was directly amplified from human genomic DNA by using PCR and inserted into vector pGEM-3Zf(+). The recombinant DNA was transformed into the host cells JM109 to obtain the positive clone pGEMBF18. The restriction enzyme analysis and DNA sequence detection confirmed that the inser ted fragment of clone pGEMBF18 is the full-length BDNF coding sequence. The hBD NF DNA fragment was recovered from the clone pGEMBF18 and ligated with prokaryot ic expression vector pGEX-5T to construct the recombinant expression plasmid p5 TBF34. The E.coli JM109 transformed with p5TBF34 was induced with IPTG. A new pr otein band with apparent molecular weight 43 kDa was detected in the lysate of t he transformed cell by using SDS-PAGE. The result of western hybridization show ed that this fusion protein reacted specifically to the antibodies to human BDNF . The amount of the soluble fusion protein was about 503.04mg/L lysate, 7.53% of total bacterial soluble protein of transformed cells, estimated by absorbance sc anning of SDS-PAGE and protein quantitation.