Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast  

作　　者：YANG Zongqi LI yinü CHEN Feng LI Dong ZHANG Zhifang LIU Yanxin ZHENG Dexian WANG Yong SHEN Guifang 

机构地区：[1]College of Life Sciences, Nankai University, Tianjin 300071, China [2]Biotechnology Research Institute, Chinese Academy of AgriculturalSciences, Beijing 100081, China [3]National Laboratory of Medical Molecular Biology, Institute of BasicMedical Sciences, Chinese Academy of Medical Sciences and PekingUnion Medical College, Beijing 100005, China

出　　处：《Chinese Science Bulletin》2006年第14期1703-1709,共7页

基　　金：supported by the National Natural Science Foundation of China(Grant No.30571189);a grant from Tianjin Municipal Science and Technology Commission(Grant No.033605111)to Yong Wang.

摘　　要：Tumor necrosis factor-related apop-tosis-inducing ligand (TRAIL) induces selectively apoptosis in various tumor cells and virus-infected cells, but rarely in normal cells. A chloroplast expres-sion vector, p64TRAIL, containing the cDNA coding for the soluble TRAIL (sTRAIL), was constructed with clpP-trnL-petB-chlL-rpl23-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant frag-ments and spectinomycin-resistant aadA gene as a select marker. The plasmid p64TRAIL was trans-ferred into the chloroplast genome of C. reinhardtii by the biolistic method. Three independently trans-formed lines were obtained by 100 mg/L spectino-mycin selection. PCR amplification, Southern blot analysis of the sTRAIL coding region DNA and culti-vation cells in the dark all showed that the exogenous DNA had been integrated into chloroplast genome of C. reinhardtii. Western blot analysis showed that human soluble TRAIL was expressed in C. reinhardtii chloroplast. The densitometric analysis of Western blot indicated that the expressed human sTRAIL protein in the chloroplasts of C. reinhardtii accounted for about 0.43%―0.67% of the total soluble proteins. These experimental results demonstrated the possi-bility of using transgenic chloroplasts of green alga as bioreactors for production of biopharmaceuticals.Tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） induces selectively apoptosis in various tumor cells and virus-infected cells, but rarely in normal cells, A chloroplast expression vector, p64TRAIL, containing the cDNA coding for the soluble TRAIL （sTRAIL）, was constructed with clpP-trnL-petB-chlL-rp123-rp12 as Chlamydomonas reinhardtii plastid homologous recombinant fragments and spectinomycin-resistant aadA gene as a select marker. The plasmid p64TRAIL was transferred into the chloroplast genome of C. reinhardtii by the biolistic method. Three independently transformed lines were obtained by 100 mg/L spectinomycin selection. PCR amplification, Southern blot analysis of the sTRAIL coding region DNA and cultivation cells in the dark all showed that the exogenous DNA had been integrated into chloroplast genome of C. reinhardtii. Western blot analysis showed that human soluble TRAIL was expressed in C. reinhardtii chloroplast. The densitometric analysis of Western blot indicated that the expressed human sTRAIL protein in the chloroplasts of C. reinhardtii accounted for about 0.43%--0.67% of the total soluble proteins. These experimental results demonstrated the possibility of using transgenic chloroplasts of green alga as bioreactors for Production of bioeharmaceuticals.

关 键 词：TRAIL 叶绿体 同源重组体 衣藻 

分 类 号：Q949.2[生物学—植物学]