Expressin Of Eriocheir Japonica Sinensis Ovarian New Gene EJO1 In E.coli BL21 And P.pastoris Gs115

Chapter 1: The current research of Eriocheir japonica sinensis ovarian new gene EJO1 and the structural genes of CrustaceaIn 2003 Ma Changyan cloned cDNA of Eriocheir japonica sinensis ovarian new gene EJO1 from the Chinese mitten crab's ovary at its developmental stage II and III with the full-length of 876 bp. The open reading frame of the cDNA consisted of 759 nucleotide encoding 252 amino acids. The molecular weight deduced from the ammo acids was 28.18 kDa.The genes which determined the structures of certain proteins (peptides) was called as structural genes. This chapter reviewed the researches about structural genes of Crustacea during the last decade.Chapter 2: Expression of Eriocheir japonica sinensis ovarian new gene EJO1 in E. Coli BL21(DE3)The recombinant plasmid pMD-18T-EJO1 was used as the template, two primers ex3591 and ex3592 were designed based on the complete sequence of EJO1's open reading frame. The complete mature peptide-coding cDNA fragment of EJO1 was obtained by PCR with the restriction sites of BamH I and Xho I. The product of PCR was cloned into the pMD-18-T to produce new construct, pMD-18T-EJO1(B/X). These fragments of EJO1 were digested with BamH I and Xho I and purified via agarose-gel electrophoresis. The ORF of EJO1 was inserted into the prokaryotic expression vector, pET28b, to produce the expression vector pET28b-EJ01. The recombinant plasmid was transformed into E.coli BL21(DE3). EJO1-HIS₆ (PRO) fusion protein was obtained after the addition of IPTG into the growth media.