CAJ | 학술논문

　　目的：构建KLK7原核表达载体,诱导hK7蛋白表达,制备抗hK7多克隆抗体。并利用此抗体研究hK7在正常前列腺,前列腺增生和前列腺癌三种组织中表达的差异及其意义。方法：PCR扩增KLK7基因,和pET-21a载体构建pET-21a-KLK7重组表达质粒。转化大肠杆菌,筛选阳性克隆,IPTG诱导表达,纯化融合蛋白并鉴定,免疫家兔获得抗血清,ELISA检测抗体的效价。免疫组化检测组织芯片中正常前列腺,前列腺增生及前列腺癌组织中hK7蛋白的表达情况。结果：成功构建了pET-21a-KLK7重组表达质粒,经转化、诱导表达和纯化,SDS-PAGE证实重组hK7蛋白与预期结果一致。免疫家兔,得到抗血清,ELISA检测抗体效价为1：200万以上。免疫组化显示在前列腺癌中的表达要比在正常前列腺及前列腺增生中的表达下降(P<0.05)。结论：获得了高纯度的hK7蛋白,及高效价的多克隆抗体,前列腺癌中hK7的表达较正常和增生前列腺组织降低,hK7可能有潜在抑制前列腺癌的作用。
　　목적：구건KLK7원핵표체재체,유도hK7단백표체,제비항hK7다극륭항체。병이용차항체연구hK7재정상전렬선,전렬선증생화전렬선암삼충조직중표체적차이급기의의。방법：PCR확증KLK7기인,화pET-21a재체구건pET-21a-KLK7중조표체질립。전화대장간균,사선양성극륭,IPTG유도표체,순화융합단백병감정,면역가토획득항혈청,ELISA검측항체적효개。면역조화검측조직심편중정상전렬선,전렬선증생급전렬선암조직중hK7단백적표체정황。결과：성공구건료pET-21a-KLK7중조표체질립,경전화、유도표체화순화,SDS-PAGE증실중조hK7단백여예기결과일치。면역가토,득도항혈청,ELISA검측항체효개위1：200만이상。면역조화현시재전렬선암중적표체요비재정상전렬선급전렬선증생중적표체하강(P<0.05)。결론：획득료고순도적hK7단백,급고효개적다극륭항체,전렬선암중hK7적표체교정상화증생전렬선조직강저,hK7가능유잠재억제전렬선암적작용。
Objective:To construct the recombinant vector,induce expression of the recombinant protein,and obtain rabbit anti-hK7 polycolonal antibody.To investigate the expression difference of human tissue Kallikreins 7(hK7)in normal prostate、BPH and prostate carcinoma.Methods:KLK7 gene was amplified by PCR.And the pET-21a-KLK7 recombinant plasmid was constructed and amplified by transformation into competent E.coli BL21 (DE3).Colony PCR was used to screen and identify positive clone.The expression of His-hK7 fusion protein was induced with IPTG. The production was purified twice and analyzed by SDS-PAGE.The antiserum was obtained by immunizing rabbits with the purified hK7 protein.The titre of antibody was detected by ELISA.The immunohistochemical assay was used to detect the expression of hK7 in Tissue Array with normal prostate tissue 18 dots,BPH 49 dots,and prostate carcinoma 26 dots. Results:The pET-21a-KLK7 recombinant plasmid was constructed successfully.After the recombinant plasmid was transformed into E. coli BL21 (DE3),His-hK7 fusion protein induced with IPTG. Purified protein was detected near 26KD by SDS-PAGE.The antiserum was obtained by immunizing 2 rabbits with the purified hK7 protein.The titer of the antibody in the rabbit serum was greater than 2,000,000. The positive expression of hK7 in prostate carcinoma was significant lower than that in normal prostate and in BPH.Conclusion:The high-purity recombinant protein hK7 and high titer antibody was successfully prepared,which will lay the foundation for the detection and research of hK7.The expression of hK7 in prostate carcinoma is lower significantly than that in normal prostate and BPH.The down-regulation of hK7 in prostate carcinoma may point out to be a possible inhibitory effect of the gene on prostatic tumar growth.