Tandem Expression Of Chicken β-defensin 5 In Escherichia Coli And Pichia Pastoris

Defensins are small molecular peptides found in many organisms which play an important role in the innate immune system.There are 14 kinds of β-defensins in chickens which have strong antibacterial activity,that can help to develop a new antimicrobial preparation.So,the successful acquisition of chicken defensins can bring social and economic benefits.However,the expression of natural defensin in chicken is low,the separation is difficult,and the synthesis cost is high.In order to obtain a large amount of active Gal-5 protein,the gene tandem technology was used to express chicken Gal-5 recombinant protein.The content and results are as follows:1.Augmentation of Gal-5 gene sequence and tandem expression in Escherichia coli.According to the c DNA coding region sequence of chicken Gal-5 in NCBI(accession number: AY621307)and E.colithe mature peptide gene of Gal-5 was optimized and the signal peptide was excised,and the 2× tandem was obtained by ligation and ligation of the peptide.The restriction endonuclease Nde I and Eco RI restriction sites were introduced into the sequence to design primers.The 2×Gal-5 mature peptide gene fragment was amplified,and the resulting vector was digested with the expression vector Pet-28 a to construct the recombinant expression vector Pet-28a-2×Gal-5,and the recombinant plasmid was transformed.The expression of E.coli BL21 was induced by IPTG.The results showed that the expression of IPTG was 0.5 m M at 20 °C for 12 h.The results of SDS-PAGE and Western Blot showed that the size of the expressed His-tagged recombinant protein was about 15 k Da.It indicated that the mature peptide sequence 2×Gal-5 was successfully expressed in E.coli,but mainly existed in the form of inclusion bodies.2.Amplification of Gal-5 gene sequence and tandem expression in Pichia pastorisAccording to the c DNA coding region sequence of chicken Gal-5 in NCBI(accession number: AY621307)and Pichia pastoris preference,the mature peptide gene of Gal-5 was optimized and the signal peptide was excised,and the 2× tandem body was obtained by first-to-tail tandem connection of the peptide.The restriction endonucleases Eco RI and Not I restriction sites were introduced into the sequence,and primers were designed to amplify the 2×Gal-5 mature peptide gene fragment.The resulting sequence was digested with the expression vector p PIC9 K to construct recombinant expression.The vector p PIC9K-2×Gal-5 was used,and the correct plasmid was linearized with restriction endonuclease Sal I,and electroporated into Pichia pastoris GS115 to induce expression with methanol.The expression supernatant was induced by SDS-PAGE and Western Blot.The results showed that Gal-5 was expressed in the supernatant of the bacterial solution after induction at 30 °C for 72 h at the methanol concentration of 1%.The expected results of SDS-PAGE and Western Blot were obtained with the bands of 12 k Da..Consistent,indicating that the mature peptide sequence 2×Gal-5 was successfully expressed in Pichia pastoris.