Cloning,Expression Of Cholecystokinin-39 (CCK39) Gene Of Pig And Preparation Of Anti-CCK39 Antibody

Cholecystokinin (CCK) is a gastrointestinal and neuropeptide that is involved in many gastrointestinal functions including gallbladder contraction, pancreatic enzyme secretion, stimulation of gastrointestinal motility and inhibition of gastric emptying. Cholecystokinin (CCK) is satiety signals to the central feeding system and inhibition of feeding. CCK is a heterogeneous protein that exists in a number of molecular forms derives from a propeptide precursor. Processing to the different forms is highly tissue specific and probably also species dependent. A large precursor peptides were prominent in gut extracts. CCK39 is one of the major molecular forms in gut of animals, it was prepared Anti-CCK antibody.Recent studies showed that food intake and growth were enhanced by immunization against CCK. To develop the plenty recombinant CCK39 and to develop special antibody, we finished following work:1. Cloning, Exprssion of Cholecystokinih-39(CCK39) Gene of PigBased on the published nucleotide sequence of pig Cholecystokinin-39(CCK39) gene, a pair of primers were designed and synthesized. Total RNA was isolated from superior duodenal flexure of pig and used as template. The DNA fragments were amplified by one-step RT-PCR and cloned into pGEX-6p-l vector. DNA sequencing confirmed that the sequence was identical to that of CCK39 published in GenBank. The recombined expression plasmid, pGEX-CCK39, was transformed into Eschorichia coil BL21 cell and induced with IPTG. SDS-PAGE confirmed that the fusion protein GST-CCK39 was about 32.0kD. The specific reactions of the fusion protein with polyclonal antibody against GST were confirmed by Western blot. The data indicated that the fusion protein has been exprssed.2. Preparation of Anti-CCK39 monoclonal antibodyThe GST-CCK39 was extracted from the gene-engineering bacteria E. coli and identified by SDS-PAGE.The gel strip containing GST-CCK39 was cut off to immunize BALB/c mice. The splenocytes of immunized mice were fused with SP2/0 myeloma cells by a routine method and the hybridomas were selected in HAT medium. The hybridoma cells secreting specific antibody were detected by ELISA, Dot-ELISA, Western blot and cloned by limiting dilution.The stability of the obtained hybridoma cells and the specificity of Anti-CCK39 monoclonal antibody (mAb) the hybridoma cells secreted were identified. One hybridoma cell line which could stably secrete specific monoclonal antibody(mAb) were called 1E7.3. Preparation of egg yolk antibody(IgY) against Cholecystokinin-39Hens were immunized with partially purified the fusion protein GST-CCK39.The titers of specific antibody to GST-CCK39 were detected by Agar Gel Precipitation Text (AGPT). One-step polyethylene glycol(PEG) precipitation procedure was used to purify the immunoglobulin Y (IgY). The titres of purified IgY were detected by Enzyme-linked. Immunosorbent Assay (ELISA) and Dot-ELISA. The AGPT titer in the IgY was higher than 1:16. The ELISA titers in the purified IgY was higher than 1:12800. High titers IgY to GST-CCK39 may be successfully induced by immunization, the specific activity lasted more than 60 days.