Tandem Expression Of AvBD2 In Escherichia Coli And Functional Analysis

Chicken ?-defensin 2?chicken beta-defensin 2,AvBD2?is a kind of cysteine-rich small peptide composing of 39 amino acids which plays an important role in the chicken's immune system because that AvBD2 has a strong killing effect on small peptides of pathogenic microorganisms.This study with chicken AvBD2 as the research object,references the preferred codons of E.coli and AvBD2 mature peptide gene of synthetic chicken,constructs the tandem expressing vector pET-28a-AvBD2op-AvBD2,transformes the constructed expression vector into Escherichia coli BL21,induced by IPTG to express the fusion protein,purified by Ni NTA purification for Western blot,Tricine SDS page electrophoresis and MALDI-TOF-MS analysis,and preliminarily studies the antibacterial activity of the purified proteins.The main contents and results are as follows: 1 The design and synthesis of mature peptide gene from chicken beta-defensin 2Referenceing the preferred codons of E.coli,in the situation of no change of the amino acid sequence,according to the chicken AvBD2 cDNA of code of sequence?nm NM₂04992?published in GenBank,rare codon of Escherichia coli is replaced for preferred codons,synthetic E.mature peptide gene sequence AvBD2 op optimized of bar fungus preference codon.Sequence at both ends respectively added BamH I and ECOR I restriction sites to construct the cloning vector pMD18-Tsimple-AvBD2op;both ends of the sequence of synthetic chicken beta defense element 2 mature peptide gene were added Not I and ECOR I restriction sites,which was cloned into pmd18 tsimple carrier after the success synthesis to construct the cloning vector pMD18-Tsimple-AvBD2.2 Tandem expression and purification of chicken beta 2 fusion proteinPMD18-Tsimple-AvBD2 op and pET-28 a were connected with BamH I and EcoR I double enzyme to construct the expression vector of pET-28a-AvBD2 op.On the basis,pET-28a-AvBD2 op and pMD18-Tsimple-AvBD2 are enzyme with Not I and Eco R I to construct the tandem expression vector pET-28a-AvBD2op-AvBD2.The recombinant plasmids were isolated and purified after enlargement culturing,and verified by PCR and gene sequence analysis.we picked up the positive transformant clone cultured to exponential phase with IPTG induced expression.The expressed product was purified by nickel column,and the concentration of protein was determined by BCA assay.By Western blot,single expression of engineering bacteria and tandem expression of engineering bacteria are at the 9500 Da have specific bands,which vertifies that the target protein AvBD2 in Escherichia coli engineering bacteria achieves a secretory expression.The protein concentration of pET-28a-AvBD2op-AvBD2 was 0.304 mg/mL after the BCA method was purified.The results of MALDI-TOF-MS analysis show that the molecular weight of the sigle expression protein products is 4339.8Da,and the molecular weight of tandem expression protein is 8385.6Da,and the molecular weight is consistent with the theory molecular weight of the protein.3 functional evaluation of chicken beta 2 fusion proteinWith agar hole bacteriostatic method to detect the expression of vitro antibacterial activity outside product.The results showed that AvBD2 fusion protein of Enterococcus faecalis Enterococcus faecalis atcc29212),Escherichia coli?E.coli CMCC44102?and Pseudomonas aeruginosa bacteria?Pseudomonas aeruginosa atcc27853?,influenza A paratyphoid salmonella?Salmonella paratyphi atcc9150?of the Pasteurella?Pasteurella?and type B paratyphoid salmonella bacteria?Salmonella Paratyphi B CMCC50094?has certain antibacterial activity.Studies have shown that we successfully constructed the tandem expressing vector pET-28a-AvBD2op-AvBD2 in this paper,using Western blot method,Tricine SDS PAGE electrophoresis and MALDI-TOF-MS analysis,which prove that the vector in Escherichia coli was successfully expressed in the AvBD2 and expression product was purified by Ni column,the purified protein by agar hole diffusion inhibition assay showd different antibacterial activity for a variety of microorganisms,which laied the foundation for the production and application of chicken beta-defensin 2 gene engineering.