The Expression Of Site-specific Modification Of Porcine GLP-2 In Lactobacillus Plantarum And Research On The Bioactivity

Piglets at weaning period due to various stress factors,can cause intestinal morphology and structure of varying degrees of damage, disorders of digestion function at weaning period, which result in the inappetence, diarrhea, growth retardation, low feed utilization rate, mental condition and poor appearance during the livestock production process. That is so-called "weaning stress of piglets syndrome" seriously affected piglets health and breeding efficiency. Glucagon-like peptide-2(GLP-2) is an enteroendocrine hormone which is uniquely trophic for the intestine,which plays a significant role in the adaptive regulation of reduced epithelial damage,intestinal cell proliferation, decreased mortality or gut injury and mucosal integrity.Native GLP-2 is rapidly cleaved by dipeptidyl peptidase IV(DPP-IV) in the second amino acid site with a short half life. Therefor, amino-acids site-specific modification has been conducted towards the porcine GLP-2(p GLP-2) genes and replaces alanine in the second site of p GLP-2 by glycine and thus forms p(Gly2)-GLP-2 which could not been digested by DPP-IV in this research. Besides, the p GLP-2 gene sequence was engineered with lactobacillus plantarum-optimied codon usage, meanwhile clone it to lactobacillus plantarum expression vector p SCPSP which expressed by exogenous gene and driven by super promoter SCP. The expression vector p SCPSP-p(Gly2)-GLP-2 constructed after the double enzyme cutting identification.Then, making electro transformation of host bacteria of lactobacillus plantarum. The positive recombinant transformant p SCPSP-p(Gly2)-GLP-2-LP1 has been achieved through identification of genome PCR method and then Tricine-SDS-PAGE was conducted to detect the interest stripe on the 24 h cultural supernatant at 4 KD. Using specific ELISA kit of p GLP-2 to detect the the expression levels of interest protein on the cultural supernatant at 24 h, 36 h and 48 h. The results showed that the expressionlevels decreased with the increase of time. The optimal incubation time being confirmed at 24 h. In order to validate the in vivo bioactivity of p(Gly2)-GLP-2expressed in this test, the chronic colitis model of mice had been constructed induced by DSS. The p(Gly2)-GLP-2 expressed that exhibited a good biological activity in the DSS-induced chronic colitis model of mice. In conclusion, The construction of the expression system composed of super promoter SCP expressed the optimied codon usage p(Gly2)-GLP-2 gene in the lactobacillus plantarum successfully and having a good bioactivity in vivo and in vitro, which provides theoretical basis and technical means for the problem of piglet weaned stress syndrome during the livestock production process.