Cloning,Exprssion And Immunogenicity Study Of Cholecystokinin-33 (CCK33) Of Pig

Cholecystokinin (CCK) is a brain-gut peptides that are synthesized byenteroendocrine I cell of dodecadactylon and jejunal.CCK exists in animal centralnervous system,intestine,peripheral blood,peripheral nerve and some tissues andorgans.As an endogenous satiety signal, CCK inhibit feeding. The biologicalfunction of CCK is stimulation of pancreatic enzyme secretion and gallbladdercontraction, the inhibition of gastric emptying, the accelaeration of insulin andsomatostation releases from pancreatic islets. Cholecystokinin (CCK)is a keyhormone to regulate animal food intake. Recent studies indicate that food intake andproduction trait was enhanced significantly by active immunity and passiveimmunity with CCK. But most studies use the CCK8 which is artificial synthesis andexpensive. Moreover, CCK8 doesn't have any immunogenicity. It must be combinedwith large molecular vector, the efficiency of crosslinking is instability, so it's hardto operate. And CCK8 combined with different vectors will have differentimmunogenicity. So far there were no report about the best vector.Constructing geneengineered protein will change this status,and become more and more popularrecently.CCK has the various molecular forms; Cholecystokinin octapeptide has thewhole biological function of CCK. CCK33 is the most stable form in the intestine. In order to get more CCK gene engineered protein which is cheap and goodimmunogenicity, and reduce the cost of research, we carried out the following work andreceive the following results:1,Designed a pair of primers according to the published nucleotide sequence ofpig cholecystokinin-33 (CCK33) gene. Use the cDNA of superior duodenal flexureof pig as template to amplify CCK33 gene which is about 100bp.2,Based on the isocaudamer enzyme existed in primers, we constructed CCK33concatemer. The CCK33 concatemer according with expect design is approximately480bp.3,The prokaryotic expression of CCK33 concatemer and the purification of thefusion protein including condition optimization were accomplished. VectorpET28a-4CCK33 was constructed and transformed into E. coli BL21 (DE3) tooverexpress the recombination protein; inclusion bodies were washed then theproteins acquired. TYPG medium was utilized. Cells were incubated with IPTG for4h at 37℃to sufficiently express the protein. Then SDS-PAGE analysis wasperformed and the outcome indicated that the protein was 16kD, which wasconsistent with anticipation. However, proteins expressed remained in the form ofinclusion bodies in the re-suspended solution after ultrasonic disruption. We washedthe inclusion bodies with relevant Buffer then acquired the fusion protein withrequired purity.4,Prepared polyclonal antibodies against the protein of CCK33 concatemer anddetected its immunogenicity and immunologic competence. Rabbits weremultisubcutaneouly injected to produce the protein-specific antibodies. Proteinsacquired through purification were utilized as antigens. And detected the change ofcontent of CCK in serum using kit. The titer of antibodies detected by ELISA was1:64000, Western blotting showed antibodies have good specificity. Next wedemonstrated CCK33 concatemer has immunogenicity.5,Constructed CCK33 gene to pGEX-4T-1 vector, express and purify GST—CCK33 protein, and detected its immunogenicity. SDS-PAGE result showed0.05mmol/L IPTG induced pGEX-CCK33/BL21 for 4h at 28℃, most GST-CCK33 protein expressed solubility form with 29KD. We purified GST-CCK33 protein withglutathion-sepharose then acquired the pure fusion protein. ELISA results showedGST-CCK33 has immunogenicity.We get Cholecystokinin proteins by different recombination technique. It provida easy and cheap way to gain the CCK gene engineered protein for the further study, andis profit to study the deffference about immunogenicity and effect among thses proteins.