Clone And Expression Of Porcine Cholecystokinin And Biological Function Study Of Cholecystokinin Regulating Pigs’ Food Intake

It has been well known that Cholecystokinin（CCK） is an appetite regulation satiety factor.Neither sulfated CCK-8 nor nonsulfated is expensive.The purpose of this topic research was studying the antigenicity of the recombinant protein of multiple concatamers of pigs’ CCK-33 expressed in vitro with genetic engineering technology and the biological function of enhancing food intake of growing-fininshing pigs supplied by this recombinant protein.In this research,the recombinant plasmid of nine concatamers of optimal pigs’ CCK-33 gene was constructed and Z9CCK recombinant protein was also expressed in BL21（DE3） Escherichia coli（E.coli）,then the antigenicity of Z9CCK recombinant protein proved out.Layers were used to immunize with Z9CCK recombinant protein and the eggs from the immunized layers were collected,and then anti-CCK egg yolk was prepared. At last,the food intake and body weight of growing-fininshing pigs were studied by making them immunized with Z9CCK recombinant protein.Results were as follows:1.The pRESET B plasmid was used as a clone vector and DH5αE.coli was clone host bacteria,and the Z9CCK recombinant plasmid was cloned;The pRESET B plasmid was also used as a expression vector and BL21（DE3） E.coli was expression host bacteria,and the Z9CCK recombinant protein was expressed.The CCK-33 bias sequences for E.coli expression system was designed and synthesized according to CCK-33 gene sequence published on the Center for Biotechnology Information （NCBI）,and the codon usage database was also considered as reference evidence.The synthesized optimal CCK-33 sequence was cloned to pRSET B plasmid through procedure of PCR,digested by restriction enzyme,ligation and transformation,and then both the CCK-33 monomer with stop codon and without stop codon was gained. On this base,recombinant plasmid of pRSET B-Z3CCK,pRSET B-Z5CCK,pRSET B-Z7CCK,and pRSET B-Z9CCK with stop codon were successfully constructed through clone for several times.Restriction of BamHⅠand BglⅡwere used for several times in cloning because they are isocaudamners.The CCK-33 multiple concatamers plasmids constructed were sequenced and the results accorded with the design for expectation.The positive clones of pRSET B-Z7CCK and pRSET B-Z9CCK were transformed into the expression bacteria of E.coli of BL21（DE3） respectively.IPTG was used as an inducer to express the recombinant protein of Z7CCK and Z9CCK.The amount of Z7CCK and Z9CCK was about 28%and 35.6% of the total protein of BL21（DE3） respectively.The expression conditions of Z9CCK recombinant protein were optimized and the results were as follows:4h was the most suitable time for induction by IPTG and Z9CCK recombinant protein expressed was not sensitive to concentration of IPTG.Therefore,amounts of CCK protein were able to provide by the way of prokaryotic expression,and the expression level of Z9CCK recombinant protein was not influenced with a low concentration of IPTG（final concentration:0.2mmol/L）.So the costs of CCK protein produced decreased.2.The prediction of antigenicity of Z9CCK recombinant protein was performed by the method of "Bepipred Linear Epitope Prediction",and then,the antigenicty of Z9CCK recombinant protein was studied with Western-blot and ELISA trial. According to the method of "Bepipred Linear Epitope Prediction" from the web site of http://www.immuneepitope.org/tools/bcell/tutorial.jsp.,the antigenicity of Z9CCK recombinant protein was predicted and the results were that two epitopes of PKAPSGR（1-6） and SLDPSHRIS（16-24） could be provided by each CCK-33 monomer.One molecular of Z9CCK recombinant protein could present 18 epitopes. We can infer that Z9CCK recombinant protein presents good antigenicty from the result of the prediction.Rabbit anti-CCK-8 polyclonal antibodies bought from Sigma corporation was used as the first antibodies and anti-rabbit IgG from goat（SBA） was used as the second antibodies,and the specialty of Z9CCK recombinant protein bound with anti-CCK-8 antibodies proved out by the assay of Western-blot.It showed that Z9CCK recombinant protein prsereved the antigenicity of CCK-8.CCK-8 synthesized by the way of chemical method（Sigma） conjugated to BSA was used as coated antigen,and anti-Z9CCK antibodies from layers immunized with Z9CCK recombinant protein purified were used as the first antibodies and mice anti-IgY antibodies were considered as the second antibodies,then it shows that anti-Z9CCK antibodies bound with CCK-8 specially by indirect ELISA assay.It also demonstrated that Z9CCK recombinant protein prsereved the antigenicity of CCK-8.So Z9CCK recombinant protein displayed good antigenicity and preserved the antigenicity of CCK-8. 3.Layers immunized with Z9CCK recombinant protein purified and the change of titers of anti-Z9CCK antibodies in eggs from layers was monitored by ELISA assay. Anti-Z9CCK egg yolk powder was prepared.750 28-week-old Hy-line brown layers were immunized with oil emulsion vaccine of Z9CCK recombinant protein.The location for immunization was breast muscle of both sides and the dosage of immunization was 0.5mL（500μg Z9CCK/mL） for each time for each location.The immune procedure was that the first immunization carried on the first day and two boosted immunization carried on 15^th and 29^th day respectively.Z9CCK recombinant protein purified was used as coated antigen and egg sample for double dilution used as the first antibodies and rice anti-IgY antibodies used the second antibodies,then the titers of anti-Z9CCK antibodies were tested.We found that CCK antibodies level boosted after one week from the first boosted immune,and the average titers of anti-Z9CCK was above 2000 and the level of anti-Z9CCK antibodies could last till 22 weeks from the first boosted immune.When the titers reached the peak of 73516.69, eggs were collected for spray-dried powder for preparation of CCK egg yolk.The average titer of anti-Z9CCK egg yolk was about 22627.42 by ELISA trial.It also demonstrated that Z9CCK recombinant protein preserved antigenicity of CCK-8.So the Z9CCK recombinant protein can be as candidate antigens used in production of egg yolk antibodies.4.Growing-fininishing pigs immunized with Z9CCK recombinant protein purified and the study of the effects of Z9CCK recombinant protein active immunization on the food intake and body weight gain of growing-fininishing pigs was carried out.72 growing-fininishing pigs（63±3d-old） of strain of Changxin were randomly divided into two treatments,9 repeats for each treatment,4 pigs for each repeat and 36 pigs for each treatment.The trial group was immunized with immunized with oil emulsion vaccine of Z9CCK recombinant protein for 2mL（500μg Z9CCK/mL）.The location for immunization was neck muscles of both sides for 1mL oil emulsion vaccine each side for one time.The control group immunized with 2mL saline instead.The immune procedure was that the first immunization carried on the first day of trial period and two boosted immunization carried on 29^th and 57^th day respectively.The results were as follow:（1） CCK antibodies could be produced effectively in the pigs of trial group immunized with Z9CCK recombinant protein. CCK-8 synthesized by the way of chemical method was used as coated antigen and it demonstrated that anti-Z9CCK antibodies could bind with CCK-8 specially;On the 29^th day,CCK antibodies were checked out in the pigs of trial group and the CCK antibodies reached peak on the 43th day.（2） During the trial period of 1-4 week,the difference of food intake and daily gain between the two treatments was not significant.During 5-6 week,the food intake and daily gain of trial group had the tendency of enhancement compared with the control group.The food intake and daily gain of trail group increased by 3.72%（P=0.10） and 5.89%（P=0.07） respectively compared with the control group.During 7-8 week,the food intake and daily gain of trail group increased by 5.02%（P=0.31） and 6.38%（P=0.34）.During 9-10 week, compared with control group,the food intake of the trial group increased by 7.22% （P=0.09） and the daily gain increased 3.80%（the difference was not significant）. During the whole period,compared with control group,the food intake of the trial group increased by 2.23%（P=0.23） and the daily gain increased 2.03%（the difference was not significant）.Therefore,the food intake and daily gain of growing-fininshing pigs could be enhanced by active immunization with Z9CCK recombinant protein.5.Growing-fininishing pigs immunized with Z9CCK recombinant protein purified and the study of the effects of Z9CCK recombinant protein active immunization on the serum hormone and blood glucose of growing-fininishing pigs was carried out by collecting the blood before food intake,during food intake and after food intake.After the feed trial about growing-finishing pigs immunized with Z9CCK recombinant protein（vide supra）,4 well healthy pigs were selected randomly from trial group and control group respectively for installing indwelling needle on the ears.At the time of 0min before food intake and 15min,30min,1h,2h after food intake,blood was collected from the 8 pigs and the concentration of CCK,insulin, blood glucose were tested with related reagent kits.The results were as follow:At the time of 1h after food intake,the concentration of CCK of trial group lowered by 4.73%（P=0.13） compared with control group;At the time of 15min after food intake, the concentration of insulin of trial group lowered by 31.62%compared with control group and the difference was significantly（P＜0.05）;At the time of 1h after food intake,the concentration of blood glucose of trial group lowered by 17.35%（P=0.19） compared with control group.It demonstrated that the concentration of CCK,insulin, blood glucose of pigs could be regulated by active immunization with Z9CCK recombinant protein,and these test results could be considered as theory basis for explaining the mechanism of food intake regulated by CCK.In conclusion,pigs’ CCK-33 mutiple recombinant protein could be expressed in E.coli successfully with genetic engineering technology and the Z9CCK recombinant protein was about 35.6%of the total protein of E.coli.Z9CCK recombinant protein displayed good antigencity and preserved the antigenicity of CCK-8.CCK antibodies could be effectively induced by making layers immunized with Z9CCK recombinant protein and be effectively accumulated in eggs.High titers of CCK yolk antibodies powder was prepared by collecting the eggs with CCK antibodies.The Z9CCK recombinant protein can be as candidate antigens used in production of egg yolk antibodies.The food intake and daily gain could be enhanced by making growing-fininshing pigs immunized with Z9CCK recombinant protein.The concentration of CCK,insulin,bood glucose of pigs could be regulated by active immunization with Z9CCK recombinant protein.