Prokaryotic Expression Of Rabbit PepT1 And Effects Of Different Ages And Different Protein Level On PepT1 MRNA Expression

There are necessary technicals mean that mRNA and protein expression of quantitative techniques to study the absorption of amino acids and PepT1 transport characteristics in the rabbits.and in this study,using Real Time PCR method determines the mRNA expression of lPepT1,CAT1,EAAC1 in different age rabbit intestinal tract and the mRNA expression of PepT1,CAT1,EAAC1 of different protein diets.Analyze the influence of age,different parts of intestinal trace,different protein diets on small peptides,basic amino acids, acidic amino acids absorption in rabbits.In order to study the absorption of small peptides from the protein level,this study establishs a model to get rabbits PepT1 protein by prokaryotic expression through genetic engineering methods for obtain protein antibodies,and it is laying the foundation for studying the absorption of small peptides in rabbits by protein level study.Trial 1 :amalyze the mRNA expression of PepT1,CAT1,EAAC1 mRNA in different age rabbits digestive.Experimental rabbits start maintain homogeneity of diets and unify free feed since wearing.Determine the mRNA expression of PepT1,CAT1,EAAC1 of the stomach,duodenum,jejunum,ileum,cecum,colon in 15,30,60,90 days by quantitative PCR method.The results show that in the the same days different intestinal segments the expression of PepTI is the highest in the ileum in each groups(P<0.05).Analyzing the expression of PepT1 among the same intestinal segments different days shows that the expression is increasing along with age in the colon(P>0.05),but the expression reachs the peak at 60 days of age in the rest of the groups(P<0.05).In the the same days different intestinal segments the expression of CAT1 mRNAis highest at 15 days of age in the stomach(P<0.05),but the expression is highest in the duodenum in the rest of the group(P<0.05).Analyzing the expression of CAT1 mRNA among the same intestinal segments different days shows that the expression is highest in the stomach at 15 days of age(P<0.05),the expression is highest in the cecum at 30 days of age,the expression is highest in the duodenum, ileum, jejunum and colon at 60 days of age(P<0.05).In the the same days different intestinal segments the expression of EAAC1 mRNAis highest at 15 and 60 days of age in the jejunum(P<0.05),the expression is highest at 30 and90 days of age in the ileum(P<0.05),the expression is relatively high in the jejunum and ileum in each group.Analyzing the mRNA expression of EAAC1 among the same intestinal segments different days shows that the expression is highest in the stomach and cecum at 15 days of age(P<0.05),the expression is highest in the ileum at 30 days of age,the expression is highest in the duodenum, jejunum and colon at 60 days of age(P<0.05).Trial 2:Different protein levels of diet effects the mRNA expression of PepT1,CAT1,EAAC1 mRNA in the rabbits digestive.According to diet recipes,experiment is divided into three groups:low protein level group,control group and high protein level group. Protein levels of diet designs gradient of 12.8%,16%,19.2%.The real measurement of protein levels is 12.4%,15.3%,17.8%.Weaning a mouth old Tarzan rabbits are divided into 3 groups(30 rabbits each group)and feed the same amount every day.The experiment lasts for 40 days.Slaughter eight rabbits each group and take intestinal tissue samples.The mRNA expression of PepT1,CAT1,EAAC1 in the duodenum,jejunum and ileum is determined by quantitative PCR method.The results shows that themRNA expression of CATI1 is highest in the duodenum in the control group,the expression tends to increase along with increasing protein level in the jejunum and ileum(P<0.05).ThemRNA expression of EAAC1 tends to increase along with increasing protein level in the duodenum and jejunum,on the contrary,the expression tends to decrease along with increasing protein level in the ileum(P>0.05)..The mRNA expression of PepT1 is highest in the duodenum, jejunum and ileum in the control group(P>0.05)..Trial 3:The purpose of This experiment is to gain the PepT1 mRNA sequenceand the PepT1 protein of rabbit by technique for PCR and gene engineering.The aim of gaining the protein is to prepare the polyclonal antibody.This experiment amplificated 2001 bp part of rabbit peptide transporter 1 gene complete sequence and 1071 bp part of rabbit peptide transporter 1 gene complete sequence which is used to prokaryotic expression successfully.The prokaryotic expression translated into a protein chain which is consisted of 357 amino acids.The results show that the gene part was cloned into the pMD18-T successfully.After gene sequencing,the recombinant pMD18-T was identified with Bamh1 and Xhol1restriction enzyme, and the Objective gene fragment were gathered by agarose gel fraction method. The fragment was ligated to the pET-32 a 、 pET-28 a express vector successfully after gene sequencing of Recombinant plasmid.pET-28a-M of prokaryotic expression vector are successfully constructed. 44 kd recombinant protein is expressed by IPTG induction. The specific approximate 44 ku band was obtained by analysis of SDS-PAGE and Western-blotting. The result shows that myostatin protein had been expressed successfully in E.coli expressing system.