AKO62675抗体的制备及其在膀胱癌中的表达研究

发布时间：2018-05-05 19:53

本文选题：膀胱癌 + 免疫组化　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:1、对前期获得的cDNA全长序列行生物信息学研究,提交NCBI获取基因号KR780251,蛋白号AKO62675。2、根据AKO62675与CK20表达差异性较大的序列设计多肽,制备AKO62675抗体:Anti-KR;3、根据AKO62675在膀胱癌组织、腺性膀胱炎以及正常膀胱组织中的相关表达情况,分析AKO62675与膀胱癌的发生发展是否具有相关性。方法:1、对cDNA全长序列进行染色体定位、蛋白分析等,并利用sequin软件向NCBI提交获取基因号:KR780251以及蛋白号:AKO62675。2、根据计算机分析AKO62675与CK20表达差异性较大的序列,以此进行多肽合成,免疫兔子并获取多克隆抗体Anti-KR。3、采用免疫组化技术对40例的膀胱癌组织、15例的腺性膀胱炎组织以及23例的正常膀胱黏膜组织行AKO62675表达的检测,对AKO62675的表达情况进行了比较,了解该片段在膀胱肿瘤组织、腺性膀胱炎组织以及正常膀胱黏膜组织间的表达情况,分析该片段与膀胱癌的发生与发展是否有关系。结果:1、分析发现该序列与CK20在染色体上所处的具体位置不同,提交NCBI获取基因号:KR780251以及蛋白号:AKO62675。2、应用ELISA方法检测成功制备出的多克隆抗体:Anti-KR,滴度1:64000,达到检测要求。3、对膀胱癌组织、腺性膀胱炎及正常膀胱组织进行免疫组化染色:在正常的膀胱黏膜中,Anti-KR染色阳性率达34.78%,而膀胱癌的组织中,Anti-KR染色阳性率达100.00%,在腺性膀胱炎组织中,Anti-KR染色阳性率也达100.00%,但染色较膀胱癌组织浅。结论:1、EST片段的全长序列KR780251有1839bp,含有完整的ORF,KR780251编码的蛋白AKO62675可能是正常膀胱组织基因的突变体。2、AKO62675在膀胱癌中具有较高的表达性。
[Abstract]:Objective to study the full-length sequence of cDNA obtained in the early stage by bioinformatics, and to submit to NCBI the gene number KR780251, protein number AKO62675.2. According to the sequence of AKO62675 and CK20, we designed the peptide to prepare AKO62675 antibody: Anti-KRR3, and then prepared AKO62675 antibody: Anti-KR3 in bladder cancer tissue according to AKO62675. The expression of AKO62675 in cystitis glandularis and normal bladder tissues was analyzed. Methods: the cDNA full-length sequence was located on chromosome, protein was analyzed, and the gene number: KR780251 and protein number: AKO62675.2 were submitted to NCBI by sequin software. According to the computer analysis of the sequences in which the expression of AKO62675 and CK20 were different, the peptide was synthesized. Rabbits were immunized and polyclonal antibody Anti-KR.3was obtained. The expression of AKO62675 was detected by immunohistochemical technique in 15 cases of cystitis glandularis and 23 cases of normal bladder mucosa, and the expression of AKO62675 was compared with that in 40 cases of bladder cancer tissue, 15 cases of cystitis glandularis and 23 cases of normal bladder mucosa. To investigate the expression of this fragment in bladder tumor tissue, cystitis glandularis tissue and normal bladder mucosa, and analyze whether the fragment is related to the occurrence and development of bladder cancer. Results: 1, the analysis found that the sequence is different from the specific position of CK20 on the chromosome. NCBI was submitted to obtain the gene number: KR780251 and the protein number: AKO62675.2. The polyclonal antibody: Anti-KR was detected by ELISA method. The titer of the polyclonal antibody was 1: 64000, which met the detection requirements. Immunohistochemical staining of cystitis glandularis and normal bladder tissue: the positive rate of Anti-KR staining was 34.78 in normal bladder mucosa, 100.00in bladder cancer tissue and Anti-KR staining in cystitis glandularis tissue. It was 100.00g, but the staining was lighter than that of bladder cancer. Conclusion the full length KR780251 of the 1: 1 EST fragment has 1839bp. the protein AKO62675 encoded by ORFN KR780251 may be a mutant of normal bladder tissue gene .2AKO62675, which is highly expressed in bladder cancer.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14

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