摘要
目的:利用毕赤酵母表达体系表达重组人腺激肽释放酶2,获得了有活性的人腺激肽释放酶2。建立测定人精浆中腺激肽释放酶2活性方法,研究测定不同类型不育症患者精浆中腺激肽释放酶活性水平。为临床相关疾病的监测,诊断和治疗提供了关键材料和方法。
方法:通过RT-PCR的方法从前列腺组织获得编码成hK2的cDNA。构建表达载体pPICZαC-mhK2及具有3个表达盒子的pPICZαC-3mhK2。采用电转法,用重组表达质粒转染毕赤酵母X33细胞,Zeocin筛选,甲醇诱导X33细胞表达rhK2,筛选高表达rhK2的细胞株。离子交换层析纯化rhK2蛋白。SDS-PAGE和Western Blot鉴定重组蛋白,使用特异性底物(H-D-Pro-Phe-Arg-pNA)检测重组hK2的酶活性。探讨了温度、pH值等对测定hK2活性的影响,设计建立测定人精浆中hK2活性的方法。
结果:获得了人hK2全长序列,构建了克隆及表达载体pPICZα-mhK2和pPICZαC-3mhK2。获得了高表达rhK2的酵母X33细胞株。离子交换层析得到纯化的rhK2,其酶活性接近正常精浆中hK2的酶活性。建立起测定人精浆中hK2活性的方法。发现多种不育症精浆中hK2的酶活性下降。
结论:毕赤酵母能够成功表达人腺激肽释放酶2,本实验获得的rhK2的酶活性接近正常精浆中hK2酶活性,我们建立起来的测定人精浆中hK2活性的方法为男性不育症的诊断提供有价值的参数,部分不育症患者精浆中的hK2活性显著下降。
Human glandular kallikrein 2 (hK2) belongs to the human kallikreinfamily of serine proteases, which comprises 15 genes located inchromosomal locus 19q13.4. KLK2 have 5 coding exons and 4intervening introns with conserved intron phase pattern. HK2 and PSAaremost homologous with a nucleotide homology between the codingregions of 80%. The expression of hK2 is regulated by androgens in aparallel fashion. hK2 is expressed mainly in epithelial cells of theprostate,The amount of hK2 found in seminal plasma is about 6 μg/ml ,only 1% of the amount of hPSA, and the hK2 concentration in normalmale serum is ~0.013 μg/L, about 2-4% of that of hPSA. hK2concentration has also been measured from other body fluids, i.e.amniotic fluid, colostrum and milk, saliva and urine. HK2 and PSAappear to be active similarly on the fragmentation of semenogelin I ,
semenogelin II and fibronectin. For fibronectin , PSA has only a lowactivity toward that substrate compared to hK2. Fibronectin is an integralcomponent of the seminal coagulum.hK2 has trypsin-like activity and cleaves high-molecular weightkininogen producing bradykinin .hK2 has been shown to activate proPSA,a zymogen form of hPSA in vitro into mature. The activity of hK2 isregulated by zinc. hK2 has been found to digest fibronectin andseminogelins I and II in seminal fluid, but not at similar positions ashPSA. hK2 has also been found to activate the single-chainurokinase-type plasminogen activator (uPA) in vitro. hK2 has beensuggested to promote the tumoral growth of prostate cancer bydegradation of insulin-like growth factor binding proteins and byincreasing the bioavailability of insulin-like growth factor.The value of hK2 in prostate cancer is under investigation.Sensitive serum hK2 assays are under development, and many in-housemethods are already in use but commercial kits are not available yet. Theexpression of hK2 is higher in cancerous prostate tissue than in benigntissue.This study aims at creating recombinant serine proteases, hK2. Inour experiments, mRNA was isolated with TRIZOL Reagent and thecDNA was gathered by reverse transcription. The cDNA encoding the fulllength human hK2 was obtained .The PCR product was inserted in TAvector, the resultant plasmid pGEM-hK2 was constructed, then thesequence of PCR product was determined. A 0.730-kb fragment wasamplified by PCR using the primer and pGEM-hK2. This production
from PCR was ligated into TA vector, the resultant plasmid pGEM-mhK2was constructed. The pGEM-mhK2 and pPICZα-C were disgested byXho I and Xba I, resulted in an inframe fusion of the mature hK2sequence with the Sacccharomyces cerevisiaeα-factor signal sequence.Bgl II and BamH I share 4 bases in common between their recognitionsites (GATC). We contructed multimers in vitro. There are 3 expressioncassettes in one multimer .The multimer was named pPICZα-3mhK2. Theresultant plasmid--pPICZα-mhK2 was linearized with the restrictionenzyme PmeI. The X33 cells were made competent as recommended bythe manufacturer's instructions, and electroporated using a BioradGenePulser. We transform multimers with uncut plasmid. Then theprotein was induced to express by methanol, and one high expression X33was chosen. The rhK2was purified from the yeast cell culture supernatantand characterized by SDS-PAGE, Western analysis, and was assayedenzyme activity by H-D-Pro-Phe-Arg-pNA. We contructed a assay toexamine the enzyme activity of hK2 from human seminal plasma. Weexamined enzyme activity of hK2 from several male infertility seminalplasma compared with human normal seminal plasma .The active, mature rhK2 was successfully in Pichia pastoris The finalyield of active rhK2 from Pichia pastoris was about 1.0mg/L. We foundmultiple copies not significantly increase the amount of protein producedcompare with the expression plasmid containing one copy of KLK2 gene .The rhK2 can be purified by anion-exchange chromatography. Theenzyme activities of hK2 from abnormal semenal plasma aresignificantly decreasing.
hK2,one of the human tissue kallikrein gene family were producedas recombinant proteins using the Pichia pastoris system. Recombinantserine proteases were purified and characterized. rhK2 presentedtrypsin-like activity. hK2 and male infertility have closerelationship.Large-scale production of active rhK2 will be useful in thethe study of hK2 biology character, clinic detection and therapeuticuse.The assay which we have contructed will be useful to diagnose maleinfertility.
引文
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