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首页> 外文期刊>Chinese science bulletin >Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast
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Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast

机译：人可溶性TRAIL在莱茵衣藻叶绿体中的表达

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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 expression 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 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 spectino-mycin 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 percent-0.67 percent of the total soluble proteins. These experimental results demonstrated the possibility of using transgenic chloroplasts of green alga as bioreactors for production of biopharmaceuticals.
机译：肿瘤坏死因子相关的凋亡诱导配体（TRAIL）选择性诱导各种肿瘤细胞和病毒感染的细胞凋亡，但在正常细胞中很少。使用clpP-trnL-petB-chlL-rpl23-rpl2作为莱茵衣藻质体同源重组片段并以抗壮观霉素aadA基因作为选择标记的叶绿体表达载体p64TRAIL，该cDNA编码可溶性TRAIL（sTRAIL）的cDNA。 。通过生物弹射法将质粒p64TRAIL转移到莱茵衣藻的叶绿体基因组中。通过100 mg / L的壮观霉素选择获得了三个独立转化的品系。 PCR扩增，sTRAIL编码区DNA的Southern印迹分析和黑暗中的培养细胞均表明外源DNA已整合入莱茵衣藻的叶绿体基因组中。 Western印迹分析表明，人可溶性TRAIL在莱茵衣藻叶绿体中表达。 Western blot的光密度分析表明，在莱茵衣藻的叶绿体中表达的人sTRAIL蛋白约占可溶性蛋白总量的0.43％-0.67％。这些实验结果证明了使用绿藻的转基因叶绿体作为生物反应器生产生物药物的可能性。

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