【作者】 王瑞；

【导师】 郑永晨；

【作者基本信息】 吉林大学 ， 临床检验诊断学， 2006， 硕士

【摘要】 CDK4分子是一种细胞周期依赖性激酶,是细胞周期G1-S期的调控中心。CDK4蛋白在Gl中后期呈表达高峰并与细胞周期蛋白D1(CyclinDl)结合成复合体而被激活,介导Rb基因产物磷酸化,从而促进细胞G1-S期的转化和细胞增殖。国内外研究证明该基因的扩增和过度表达广泛存在于人类的多种肿瘤组织及其细胞中, CDK4基因的异常与多种肿瘤的发生密切相关。为了以CDK4为突破点寻找肿瘤的诊断、鉴别诊断及抗肿瘤治疗的新途径,该分子的研究已成为一个新的研究热点。本研究从肝癌组织中获得总RNA,逆转录PCR反应获得人CDK4基因,将目的基因插入原核表达载体pET28a(+)的启动子下游,构建了原核表达质粒pET28a(+)-CDK4。将重组质粒pET28a(+)-CDK4用氯化钙法转化入宿主菌BL21(DE3)中,筛选出含重组质粒的基因工程菌。工程菌经IPTG诱导表达和SDS-PAGE电泳分析及Western blotting鉴定表明含有人pET28a(+)-CDK4重组质粒的工程菌BL21(DE3)可以表达出34kd左右的CDK4蛋白质。本研究的成果可以进一步研究CDK4的生物活性,筛选CDK4的配体和阻断剂以及制备单克隆抗体。更多还原

【Abstract】 CDK4 is an important member of CDK family, which plays a key role inregulating cell cycle. It combines with cyclin D1 and propels the cell cyclethrough the G1 checkpoint, which a critical phase of cell proliferation. CDK4can modulate cell cycle progression from G1 to S phase, facilitate cellproliferation, accelerate cell division and lead to tumorigenesis and progress.The amplification and overexpression of CDK4 gene in many tumor tissueand tumor cells imply this gene is closely related to many tumors. In order tofind new path of tumor diagnosis, differential diagnosis and antineoplastictherapy, research about CDK4 has been a new point.Human CDK4 gene locates in the 12th chromosome, has 912 base pairsin cDNA and products a polypeptide chain including 303 amino acids with34Kd. CDK4 protein is a member of the Ser/Thr protein kinase family and itsactivation involves both association with cyclinD1 and phosphorylation of aconserved threonine residue excited by Cdk-activating kinase (CAK). Theternary complex of cyclinD1/CDK4/P21 in mid-G1 phase, under effect ofCAK, stimulate the activity of CDK4, which is responsible for thephosphorylation of retinoblastoma gene product (Rb) and the release of E2Ffactor, and then propels the cell cycle alteration of G1-S phase and cellproliferation. In the cell progression, fortification of provocative element(CylinD1) and attenuation of inhibitor (Cyclin-dependent kinase Inhibitors,CKIs) may lead to cell proliferation and division, ultimately result incanceration.Many researches have confirmed amplification and overexpression ofCDK4 gene in hepatocelluar carcinoma, malignant glial tumor, malignantmelanoma, breast cancer, skin cancer, epithelial ovarian tumor, and so on. Atsame time, the inhibition of gene knockout, siRNA targeting CDK4 andCDK4 inhibitor on many tumor tissues or tumor cells is obvious. So CDK4gene is an important target of antineoplaston, and it is essential to establish amodel of selecting antineoplaston aiming directly at CDK4. Althoughfrequencies of cells in division are more than in normal cells, the content islittle. So it is difficult to abstract many CDK4 active complexes for screeningantineoplastic medicine. In this study, we cloned human CDK4 gene fromhepatoma, constructed prokaryote expression plasmid and induced CDK4protein expression. This protein can be used to further study bioactivity ofCDK4, screen suppressor and prepare monoclonal antibody of CDK4.Although DNA recombination technique can get amount of target geneproducts, the high yields of foreign gene expression are correlated to not onlythe relationship between host strain, vector and target gene, but also promoter,self-character of gene, growing temperature, concentration of inducer (IPTG)and base sequence around initiation codon.In this experiment, we designed a pair of specific primer according toCDK4 cDNA sequence and obtained human CDK4 gene from hepatoma tissuewith RT-PCR method. Sequencing analysis following TA clone indicated thegene is identical with that in Genebank (NM00075) and the CDK4 gene clonewas successful. Subsequently, we insert CDK4 gene into the downstream ofT7 promotor of prokaryotic expression vector pET28a(+) and successfulconstruct prokaryotic expression recombinant plasmid pET28a(+)-CDK4judged by double restriction enzymes digestion.The use of E.coli for protein expression is well documented for itsadvantages of low cost, easy transformation and high protein yields, whileeukaryotic expression system is strict with cell culture condition and itsexpression level is low, cost is also high. So in this experiment, we usedprokaryotic expression system pET28a (+) to express CDK4 protein. VectorpET28a (+) is one of prokaryotic expression series in Novagen company withmany characters: 1) A kana antibiotic resistance selectable marker. 2) Thenucleotide sequences in translation initiation and the distance between SDsequence and AUG are mostly optimized. 3) It has powerful T7 promoter,terminator and multiple cloning sites. 4) The downstream and upstream ofmultiple cloning sites respectively is His-tag sequence which can fuse totarget gene and express fusion protein include 6 histidines. Because 6continual histidines can reversibly combine with Ni with high specificity, sowe can purify the protein with affinity chromatograph.Obtaining target protein with genetic engineering required not onlyexcellent expression system but also corresponding host bacteria. We selectedEscherichia coli BL21(DE3), the widely used host bacteria according topET28-a(+) in this experiment. In this bacterium, there are mutation of ompTgene coding tunica adventitia protease which can degrade outer membraneprotein and Lon gene coding Lon protease which can degrade theuncomplicated unfolding protein and foreign protein. Thus, expression inBL21 (DE3) is benefit for higher yields of intact recombinant protein.In this experiment, we transformed the recombinant plasmidpET28a(+)-CDK4 into Escherichia coli BL21 (DE3) with CaCl2 methodfollowed by selecting and identifying clonal bacteria, inducing expression byIPTG, identifying protein expression with SDS-PAGE electrophoresis andWestern blotting. Result display that we successfully obtain CDK4 protein.This protein can be used to further study bioactivity of CDK4, select itsligands and series inhibitors, prepare antibody and subsequently studyantineoplastic effect. The target of medicine selected by this method is definite,and it can shorten obviously study process of medicine. The result of ourstudy makes it is possible to built specific and sensitive model of selectingmedicine and provides new path for tumor gene diagnosis and differentialdiagnosis and antineoplastic therapy.更多还原