Study On The Prokaryotic Expression Of Trypsinogen Gene And Distribution In The Yellow Broilers

Objective:Trypsinogen plays a key role in the protein digestion.In recent years,studies had found that trypsinogen not only had the digestive function,it also had other functions.There was still no detailed report about trypsinogen expression and distribution in poultry.It is significant to study on the trypsinogen in broiler for improving the production performance of broilers.Method:(1)TRY gene of yellow broiler was cloned by the cloning technology.The structure and function of TRY gene was analyzed and predicted by bioinformatics softwares.(2)TRY gene was expressed by prokaryotic expression technique.The recombinational protein was purificated and immunized in New Zealand white rabbits to prepare rabbit anti TRY polyclonal antibodies.(3)The distribution of TRY gene in yellow broiler was analyzed by immunohistochemistry,reverse transcription PCR and real-time PCR methods respectively.Then the expression of TRY gene in pancreatic tissue during different growth stages was studied also by immunohistochemistry,reverse transcription PCR and real-time PCR methods respectively.Results:TRY gene of yellow broiler was successfully cloned.Results showed that full-length of open reading frame of TRY gene was 747bp,which encoded 248 amino acids.TRY has a signal peptide of 15 amino acids(MKFLVLVAFLGVAVA)and an activation peptide of 10 amino acids(FPISDEDDDK).The molecular weight of TRY was 26.1 ku,with 11 phosphorylation sites but no glycosylation site.TRY was hydrophobic without transmembrane domain.Alignment analysis based on the amino acid sequences revealed that TRY from yellow broiler was highly conservative,which included the catalytic triad(His-64,Asp-110 and Ser-203),with 12 cysteines forming 6 disulfide bonds.Homolog analysis indicated that from yellow broiler and purebred chicken had the highest homology at 98.8%.Results showed that prokaryotic expression vector of TRY gene was successfully constructed.The recombinational protein was successfully induced in BL21 competent cells.Results showed that the recombinant protein was successfully expressed by SDS-PAGE.The optimal inducing condition was for 24h at 37?,200r/min,IPTG=0.8mmol/L.The target protein was purified by affinity chromatography with 160mMol imidazole elution.The distribution of TRY gene in yellow broiler was investigated by immunohistochemistry,reverse transcription PCR and real-time PCR methods.Results showed that male and female yellow feather broilers had similar distribution characteristics.TRY gene was distributed in the digestive system,nervous system,immune system and reproductive system,with the highest expression of pancreas.The distribution of TRY gene was extensive.And the distribution of TRY gene was different between female and male,even in different tissues.The relationship between TRY gene and the development stages of yellow broiler was researched by immunohistochemistry,reverse transcription PCR and real-time PCR methods.Results showed that male and female yellow feather broilers had similar expression characteristics.TRY gene began to express at 1stay old age.The expression of TRY gene increased with the development stages,which was significantly increased at 14th day old.The expression of TRY gene was different between male and female.After 14th day,the expression level of male yellow broiler was higher than that of female.At 14th day,the difference was significant(P<0.05).At 35th day and 56th day,the differences were extremely significant.Conclusion:1.TRY gene of yellow broiler was successfully cloned,and the TRY protein was obtained in vitro and was purified,then rabbit anti TRY polyclonal antibody was prepared.2.TRY gene of yellow broiler was distributed in the digestive system,nervous system,immune system,reproductive system,with the highest expression level in the pancreatic.3.TRY gene began to express at 1st day old in broiler.The expression of TRY gene increased with development stages.4.The expression of TRY gene showed differentiation in different sex.After 14th day old,the expression level of male yellow broiler was higher than that of female.