科研之友
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摘要
Atsttrin is a truncated mutant of granular protein precursors (Progranulin,PGRN),and it has a stronger TNFα receptor antagonism than PGRN,which can produce more significant inflammatory therapeutic effect.In order to screen out the high expression of Atsttrin prokaryotic expression system and lay a working foundation for the development of new anti-inflammatory drugs,this study was carried out.In this study using the cDNA of HUVEC cells as PCR templates,the gene of pgrn was amplified by PCR,and the coding DNA of atsttrin was then obtained by the method of overlap PCR and cloned into three different expression vectors (pET-22b,pET- 40b and pGEX- 6p-3).Then recombinant plasmids were transformed into E.coli BL21 (DE3) and Rosetta (DE3) host strain and six transformants were gained.It demonstrated that the BL21 (DE3)/pGEX-Atsttrin had higher expression of target protein.To achieve a higher expression level,a series of factor experiments were employed to optimize the expression conditions,in terms of temperature,induction time,concentration of lactose and post-induction time.The results showed that when the culture was propagated (37 ℃,200 r/min) to an optical density (A600) of 1.0,then induced by 3.0 mmol/L lactose at 30 ℃ for 12 h and under the conditions of 5 L fermenter,the recombinant protein yield was 550 mg/L after 10 h induction.Atsttrin's E.coli expression system was successfully established and its expression conditions were optimized,which laid a foundation for the development of related drugs. ? 2019, Editorial Board of Pharmaceutical Biotechnology. All right reserved.
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