【作者】 徐文华；

【导师】 乔代蓉；

【作者基本信息】 四川大学 ， 微生物学， 2007， 硕士

【摘要】 胆囊收缩素(cholecystokinin，CCK)，是一种由十二指肠和空肠的内分泌细胞Ⅰ细胞分泌的脑肠肽，广泛分布于动物的中枢神经系统、肠道、外周血液、外周神经及某些组织器官中。CCK作为一种内源性饱感激素，具有收缩胆囊、促进胰酶分泌、抑制胃排空、促进胰岛素释放等生物学功能，因此在调节动物摄食量方面发挥重要的作用。国内外广泛采用免疫调控技术来降低动物内源性CCK的含量，从而达到增加动物摄食量，提高动物生产性能的目的。在已有的研究中多数采用人工合成的CCK8作为研究材料，研究成本较高。CCK8分子量小，通常需将其与大分子载体交联后才具有免疫原性，交联效率不稳定，增加了研究的难度。而且交联载体不同，免疫效果也有差异，对于最佳交联载体的选择目前没有明确的报道。采用分子生物学的方法，构建基因工程蛋白可以改变这一现状，并且已经逐步成为目前研究的热点。CCK的分子形式具有多样性，CCK8被公认为是CCK的外周和中枢生物学活性的最小单位。CCK33是存在于肠道粘膜中的最稳定的分子形式，适用于体外重组蛋白的构建与表达。为得到大量的来源广泛、价格低廉、免疫原性良好的猪源胆囊收缩素基因工程蛋白，使得在进一步的深入研究工作中，能够大幅度的降低研究成本，本论文开展了以下的工作并取得了阶段性的成果：1．克隆了猪源胆囊收缩素33肽(CCK33)基因。提取猪十二指肠肠粘膜总RNA，并以反转录的cDNA为模版，设计特异引物成功克隆出与预期相符的100bp左右的CCK33基因。2．成功利用T载体构建了CCK33基因同向4串联体。利用NheI和XbaI的同尾酶特性及T载体上的单一酶切位点NdeI，成功构建了与预期设计相一致的480bp左右的CCK33同向4串联体，使CCK33基因达到免疫原的要求。并且此种串联方法并不局限于某个特定的基因，作为一种通用的手段，只需要稍加改造就可以适用于其他基因。3．构建了CCK33基因4串联体原核表达载体，并在大肠杆菌表达系统中获得成功表达，同时完成了相应的蛋白质纯化及相关条件的优化。构建pET28a-4CCK33重组表达载体；将其转入E．coli BL21(DE3)进行重组蛋白的表达，实验确立使用TYPG培养基，在1mmol／L的IPTG终浓度下37℃诱导表达4h，SDS—PAGE分析证实产物大小为16kD左右，与目的蛋白大小相符；目的蛋白以不溶性的包涵体形式出现在细胞破碎后的沉淀重悬液中，通过不同的洗涤缓冲液对包涵体进行反复洗涤，最终纯化得到纯净的CCK33基因4串联体融合蛋白。4．制备了CCK33基因4串联体的多克隆抗体并检测其免疫原性。用纯化得到的目的蛋白做为抗原，采用皮下多点注射方法免疫新西兰白兔，制备到目的蛋白的特异性多克隆抗体，通过ELISA测定效价为1：64000；Western杂交显示抗体特异性好，并同时证明了CCK33基因4串联体具有免疫原性。5．采用融合蛋白表达策略构建pGEX—CCK33原核表达载体，并获得成功表达，优化诱导提纯条件，获得了纯净的GST—CCK33蛋白，并对其免疫原性进行检测。将CCK33单基因构建入pGEX-4T-1表达载体，实验结果表明在0.05mmol／L的IPTG终浓度下37℃诱导表达4h；得到大小约为29kD左右的可溶性GST—CCK33蛋白，通过谷胱甘肽琼脂糖小珠纯化得到纯净的目的蛋白，ELISA结果表明GST—CCK33可与抗CCK33基因4串联体蛋白的抗体结合，表明GST—CCK33具有一定的免疫原性。总之，本论文从不同的构建策略出发，分别得到了不同的猪源胆囊收缩素蛋白，即CCK33四串联体重组蛋白与GST—CCK33融合蛋白，为以后进行CCK各种生理功能的研究提供了来源广泛价格低廉的研究材料，节约了研究成本。同时对研究不同重组形式的基因工程蛋白免疫原性之间的差异及其在动物体内作用效果的差异提供了前提，为开展胆囊收缩素蛋白大规模应用于动物生产的可行性研究奠定了基础。更多还原

【Abstract】 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.更多还原