Construction And Function Evaluation Of Chicken AvBD6in Lactic Acid Bacteria

Chicken β-defensin is an important kind of antimicrobial peptides, which has a broad-spectrum antibacterial activity, a special antibacterial mechanism, not easy to produce drug resistance to bacteria and any other characters, has a broad application prospects as a new antibiotic drugs. Based on the sequence of AvBD6mature peptide gene, considered L. lactis codon bias, a new AvBD6mature peptide gene was designed and synthesized. The expression vectors pNZ8148-Op-AvBD6were constructed and then the objective protein AvBD6was induced with Nisin. The antibacterial activity in vitro and in vivo of objective protein AvBD6was preliminarily investigated. The specific contents and results were showed as follows:1The construction of expression vector of Chicken AvBD6Landing the Genbank and searching for the cDNA sequence of chicken AvBD6（accession number:NM₀01001193）, and then adding the restriction enzyme cutting sites of Noc Ⅰ and Xba Ⅰ at each end of the AvBD6mature peptide gene sequence, a new AvBD6mature peptide gene（Op-AvBD6） was designed and synthesized after considering L. lactis codon bias, which was cloned in the vector pMD18-Tsimple. The Op-AvBD6gene was double digested and inserted into the pNZ8148vector which was also double digestted, the recombinant plasmid pNZ8148-Op-AvBD6was transformed into L. lactis NZ9000cells. The recombinant plasmid was isolated and purified after culturing, and verified by PCR, double digestion and gene sequence analysis.2Expression and antibacterial activity of AvBD6proteinThe positive clone was induced to express target protein at30℃by addition of5ng/mL Nisin for4h. Expression product whose molecular weight was approximately5kDa was examined by Tricine-SDS-PAGE, which was consistent with the actual results. The recombinant of L. lactis was broken by ultrasonic and lysozyme, and then the vitro antimicrobial activity of the target protein was determined by monlayer agar plate diffusion method, which exhibited antimicrobial activity against Escherichia coli （CMCC44102）, Pasteurella multocida, Enterococcus faecalis （ATCC29212）, Salmonella paratyphi （ATCC9150） and Salmonella pullorum （S2）.3The application of recombinant L. lactis NZ9000With chick as object, they were divided into four groups, group I was Lactobacillus group, group Ⅱ was L. lactis NZ9000empty vector group, group Ⅲ was recombinant L. lactis NZ9000group which expressed AvBD6protein, group Ⅳ was control group. A preliminary study on the effect of different groups to chicks, which was in average daily weight gain, immune organ index, intestinal flora and the preventive effect on chicks who were infected with avian pathogenic Salmonella paratyphi ATCC9150. The results showed that, group Ⅰ and group Ⅲ could improve the average daily weight gain of chicks, while group Ⅲ improved more outstandingly, at the same time, group Ⅲ could maintain the structure of intestinal bacteria stability. After being infected, group IV all died, while group I, Ⅱ and Ⅲ had survived, and the mortality rate of Ⅲ group was the lowest. It showed that recombinant L. lactis NZ9000which expressed AvBD6protein had a good prevention effect in vivo.In summary, this study constructed expression vector pNZ8148-AvBD6successfully and then transformed into L.lactis NZ9000cells. It was induced at30℃by Nisin. Tricine-SDS-PAGE detected the molecular weight of target protein AvBD6was about5kDa and it had a good biological function.