人类精子表面附睾蛋白激酶抑制剂Eppin和精囊凝固蛋白Semenogelin的相互作用研究

目的:探讨人类精子表面一种附睾蛋白激酶抑制剂Epp in和精囊凝固蛋白Sem enogelin(Sg)的相互关系。方法:①用抗Epp in的抗体与精子和精浆中Epp in蛋白进行免疫沉淀反应;②免疫荧光法在精浆和精子表面共同显色定位;③重组Epp in(rEpp in)和重组精囊凝固蛋白Sg(rSg)共同孵育,用抗Epp in抗体或抗Sg抗体进行免疫共沉淀;④W estern印迹检测rEpp in和rSg相互作用;⑤放射性125I-rSg与rEpp in结合达饱和后,用未标记的rSg竞争性结合分析;⑥放射性125I-rSg直接与印迹膜上的rEpp in行放射性自显影。结果:人精子表面蛋白Epp in与精囊蛋白Sg发生特异性结合,Sg分子结构中的半胱氨酸残基Cys-239是唯一的结合位点,Cys-239的还原及羧甲基化将阻碍125I-rEpp in与rSg的结合。结论:Epp in与Sg的结合是靠分子结构中二硫化键的参与。在人类射精后的精子表面,精囊蛋白Sg结合在精子表面的Epp in上。     

重组人前列腺特异性抗原的原核表达、纯化与鉴定

目的:用分子克隆技术体外制备人前列腺特异性抗原(PSA)重组蛋白,并对其活性进行鉴定。方法:用分子克隆技术从前列腺癌cDNA文库中扩增PSAcDNA,再将cDNA和准备插入的质粒载体PET-12α分别用限制性内切酶NdeⅠ和BamHⅠ消化,然后用T4连接酶将两者连接,序列正确的阳性克隆转染BL21(DE3)大肠埃希菌,诱导表达重组人PSA蛋白,从细菌包涵体中纯化PSA蛋白,再用小剂量胰岛素激活PSA活性,观察活化的PSA是否分解其变色底物S-2586和天然底物精囊蛋白Semenogelin(Sg)。结果:利用原核表达技术得到了重组人PSA,在胰岛素作用下PSA被激活,活性PSA水解其天然底物Sg和变色底物S-2586。结论:用基因克隆方法体外获得的重组人PSA蛋白可表现与天然PSA同样的丝氨酸蛋白酶活性和功能。     

附睾蛋白酶抑制剂对前列腺特异性抗原活性的抑制作用

目的 观察附睾蛋白酶抑制剂(Eppin)是否抑制前列腺特异性抗原(PSA)酶活性.方法 利用分子克隆技术体外构建、表达、纯化重组Eppin和PSA.利用PSA水解变色底物S-2586的颜色变化,于分光光度计下测定其吸光度值,代表PSA酶反应速度,反应体系是在缓冲液0.1 mol/LTris-HCl,pH8.3,1.0 mol/L NaCl中进行,观察不同浓度Eppin的加入对PSA酶反应速度的影响.结果通过体外重组技术获得较高纯度和生理活性的Eppin和PSA,通过PSA水解变色底物S-2586的检测,重组PSA具有酶活性,0.3 μmol/L PSA与底物S-2586反应的饱和曲线分析显示Km(反应速度为最大反应速度一半时的底物浓度)值是0.5 μmol,底物饱和浓度0.2 mmol/L,分别加入4中不同浓度的重组Eppin 0、0.56、1.16、2.32 μmol/L,随着Eppin浓度的增加,PSA水解其变色底物S-2586速度明显减慢,PSA活性越来越受到抑制.结论 附睾蛋白酶抑制剂(Eppin)是前列腺特异性抗原(PSA)一种新的特异性抑制剂.

Abstract：

Objective To investigate the inhibitory effects of the epididymal protease inhibitor (Eppin) on the activity of prostate specific antigen (PSA).Methods Recombinant Eppin and recombinant PSA were produced by molecular cloning technique in vitro.The enzymatical analysis of Eppin inhibiting PSA was done in the reaction buffer 0.1 mol/L Tris-HC1,pH 8.3,1.0 mol/L NaCl.The hydrolysis of velocity of PSA to the chromogenic substrate S-2586 was detected by spectrometer.Results Recombinant Eppin and PSA with high purity were produced by molecular cloning technique.The recombinant PSA had the enzymatical activity by hydrolyzing its substrate S-2586.0.3μmol/L PSA and the substate S-2586 reaction to the saturation curve analysis showed that Km( response rate of half the maximum reaction rate when the substrate concentration) value was 0.5μmol,and substrate saturated concentration was 0.2 mmol/L.With the increases of Eppin (0,0.56,1.16,2.32μmol/L),the hydrolysis velocity of PSA to S-2586 was slowed down.Conclusion Eppin,as a new inhibitor,specifically inhibits the activity of PSA.     

精囊蛋白Semenogelin抑制精子活动的活性片段分析

目的:制备重组Sem enogelin(Sg)及其不同的氨基酸片段,研究其对精子活动的影响。方法:设计特异性的引物,用分子克隆技术从精囊cDNA文库中扩增精囊蛋白Sg及其N端和C端片段的DNA,再将DNA插入质粒载体PET100,筛选序列正确的阳性克隆,转染BL21(DE3)大肠埃希菌,诱导表达重组人类Sg蛋白及其N端和C端片段,用50%N i-NTA纯化重组蛋白,用上游法筛选出的正常生育男性活动精子行抑制分析,研究重组Sg及其两片段在4种不同浓度(0、1、5、10)ng/μl下对精子活动的影响。结果:重组Sg及其N端片段在5、10 ng/μl浓度下明显抑制精子的运动(P<0.001),C端片段对精子运动无抑制作用(P>0.05)。结论:Sg分子中明显抑制精子运动的活性片段在氨基端,精液液化过程中,必须清除这端抑制片段,精子才能前向运动。     

附睾蛋白酶抑制剂Eppin的研究进展

附睾蛋白酶抑制剂Eppin是一种睾丸和附睾特异性分泌的蛋白质,精液中含量极为丰富,精子表面大量存在,用重组Eppin免疫的猴子出现不育,Eppin有望成为人类有效的可逆性的免疫避孕疫苗。现就Eppin基因及其蛋白质结构功能特点,Eppin引起免疫性不育的分子机制以及Eppin调控PSA水解精囊蛋白Semenogelin的研究进展进行了综述。     

Binding of the carboxyl-terminal fragments of human parathyroid hormone to rat osteoblastic cells ROS 17/2.8

We have recently demonstrated that the carboxyl-terminal (C-terminal) PTH fragments increase or decrease alkaline phosphatase activity in dexamethasone-treated ROS 17/2.8 cells, depending on the length of deletion of amino-terminal amino acids of the PTH molecule, and interact with amino-terminal (N-terminal) PTH fragment [Acta Endocrinol 128:367]. In the present study, we examined individual and combined inhibitory effects of N-terminal and a series of truncated C-terminal PTH fragments [PTH (1-34), (35-84), (53-84) and (71-84)] on the binding of intact PTH molecule [PTH (1-84)] to ROS 17/2.8 cells. The C-terminal PTH fragments, as well as the N-terminal PTH fragment, partially inhibited the binding of [³⁵S]-labeled PTH (1-84) to the cells. The inhibitory effect of C-terminal PTH fragments was reduced along with the deletion of aminoterminal amino acids of the PTH molecule, but still retained in the shortest C-terminal PTH fragment, PTH (71-84). When added together, PTH (1-34) reinforced the inhibitory effect of each C-terminal PTH fragment. The combination of PTH (1-34) and the complementary C-terminal PTH fragment, PTH (35-84), resulted in inhibition of [³⁵S] PTH (1-84) binding to the level obtained by addition of the same concentration of unlabeled PTH (1-84).     

人血管内皮生长因子C基因真核表达载体的构建

目的：VEGF—C基因的真核表达载体，以便进一步研究VEGF—C基因在淋巴管生成中的作用。方法：根据人VEGF—C的cDNA序列，设计合成一对5’端分别含有EcoRI和BamH I酶切位点的特异性引物，运用RT—RCR方法扩塔人乳腺癌细胞MDA—MB－231中的VEGF—C cDNA(1.26kb)；回收PCR产物(1．28kb)，并将其连接至克隆载体pUMT—18中，重组的pUMT—18在大肠杆菌DH5α内扩增后，经质粒提取、EcoR I和BamH I酶切，筛选出阳性重组子，并进行基因测序鉴定；琼脂糖凝胶电泳回收含有VEGF—C cDNA全长的酶切片断(1．27kb)，然后在DNA连接酶作用下将其与真核表达载体pcDNA3．1（－)连接，重组质粒经EcoR I和BamH I酶切予以鉴定。结果：RT—PCR产物自有VEGF-C cDNA，基因测序显示重组的pUMT—18中自有正确的人VEGF—C cDNA全长序列，重组的pcDNA3．1(-)中含有人VEGF—C cDNA全长序列。结论：VEGP—C/pcDNA3．1(－)。     

Molecular heterogeneity of free PSA in sera of patients with benign and malignant prostate tumors.

OBJECTIVE: To analyze free prostate-specific antigen (f-PSA) in sera from patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH), and to detect possible differences in subtypes as potential diagnostic parameters. MATERIALS AND METHODS: PSA was purified from sera by an immunoaffinity procedure developed on the basis of oriented antibody immobilization, and subjected to size exclusion chromatography (SEC), Western blotting, and N-terminal amino acid sequencing. RESULTS: The novel procedure allowed the purification of PSA with high yield from sera containing PSA <10 ng/ml. SEC under nonreducing conditions as well as Western blots demonstrated the presence of several molecular forms of f-PSA. Three of the smaller polypeptides exhibited the N-terminal sequence of PSA while one represented the C-terminal fragment Lys(146)-Pro(237). Shortening of some polypeptides by the N-terminal amino acid Ile(1) suggestive of aminopeptidase action was also observed. No propeptide sequence could be detected, and none of the bands from patient sera reacted with antibodies raised against propeptide antigens. BPH sera expressed higher proportions of smaller PSA fragments per unit p33, and contained significant amounts of fragments <14,000 which appeared to be very low or absent from most PCa sera. CONCLUSIONS: f-PSA as obtained from BPH and PCa sera represents a heterogeneous fraction. The major component (p33) is not in the nicked form and does not contain proPSA. Diagnostic potential could arise from the quantitative differences of the smaller PSA derivatives seen between PCa and BPH sera.     

Quantification of seminal plasma motility inhibitor/semenogelin in human seminal plasma

Semenogelin I and II (Sg I and II) are the major components of human semen coagulum. The protein is rapidly cleaved after ejaculation by the chymotrypsin-like protease prostate-specific antigen (PSA), which results in the liquefaction of the semen coagulum and the progressive release of motile spermatozoa. One of the cleavage products of the protein, a 14-kDa protein, is a sperm motility inhibitor (seminal plasma motility inhibitor [SPMI]). We developed a monoclonal antibody (mAb) that is specific to the fragment of Sgs, SPMI, and a sandwich enzyme-linked immunosorbent assay (ELISA) system for the quantification of Sgs using this mAb. Then, we measured SPMI/Sg levels in human seminal plasma from healthy male volunteers (n = 100, aged 18-24 years). The mean level of SPMI/Sg in seminal plasma was 19 +/- 13 mg/mL (range, 4-68 mg/mL). Log-transformed SPMI/Sg levels were negatively correlated with the sperm motility (r = -0.229, P =.0220) and positively correlated with the total protein concentration (r = 0.793, P <.0001). This result supports that SPMI, one of the fragments of Sg, has its inhibitory effect on ejaculated spermatozoa in liquefied semen under physiological conditions.     

Semenogelin, the main protein of semen coagulum, inhibits human sperm capacitation by interfering with the superoxide anion generated during this process.

Semenogelin (Sg), the major protein of the human semen coagulum, is present at high concentrations in seminal vesicle secretions. It is degraded by the prostate-specific antigen (PSA) to generate peptides of various biological activities that were found on and inside spermatozoa. Our aim was to determine the effect of Sg on capacitation, which is the series of transformations that spermatozoa must undergo to become fertile. At concentrations of 0.1 to 1.0 mg/mL (600- to 20-fold lower than those of semen), Sg did not affect sperm motility (%) but completely prevented capacitation induced by fetal cord serum ultrafiltrate; a partial inhibition of capacitation was noted with 0.03 mg Sg/mL. There was also a dose-dependent decrease in the tyrosine phosphorylation of fibrous sheath proteins and in the O2-.-related chemiluminescence. Ribonuclease (RNase), which has as high an isoelectric point (pI = 9.7) as Sg (pI = 9.5), also prevented sperm capacitation and O2-.-related chemiluminescence but to a lower extent, suggesting that one mechanism of Sg action on spermatozoa could be related to its positive charge at physiological pH. Sg at 1, but not 0.3 or 0.1 mg/mL, scavenged the O2-. generated by the mix of xanthine + xanthine oxidase and modified the kinetics of the reaction; RNase did not have such effects. Therefore, Sg is a potential scavenger for O2-. but probably also affects the sperm oxidase. Spermatozoa rapidly processed Sg; a high proportion of Sg was degraded after 15 minutes of incubation. The resulting polypeptide patterns were reminiscent of those obtained with PSA as a proteolytic enzyme. These data suggest that Sg, its degradation products, or both may be natural regulators of sperm capacitation and could prevent this process from occurring prematurely. One mechanism by which Sg acts could involve an interference with the O2-. that is normally generated during this process.     

大鼠Uqcrfs1重组质粒的构建及其在HEK293T细胞中的表达

目的:构建大鼠Uqcrfs1的重组质粒并检测其在人胚胎肾293T细胞中的表达。方法:应用RT-PCR方法从大鼠肾脏组织总RNA中扩增出编码Uqcrfs1的cDNA,克隆至pUM-T载体并测序,然后亚克隆至真核表达载体pcDNA3.1/V5-His,酶切鉴定及测序正确后以磷酸钙共沉淀法瞬时转染HEK 293T细胞,使用RT-PCR、Western Blot方法检测重组质粒在转录及蛋白水平的表达。结果:测序结果证实PCR扩增得到编码Uqcrfs1的cDNA序列正确;磷酸钙共沉淀法转染HEK293T细胞后,在基因转录与蛋白表达水平,结果达到预期。结论:成功构建大鼠Uqcrfs1的重组质粒,该重组质粒可在HEK293T中过表达。     

鼠肌肉转录调节因子MyoD基因真核表达载体的构建

目的 构建肌肉转录调节因子MyoD基因真核表达载体,为深入研究MyoD在肌肉修复中的分子调节机制,探讨其在肌肉损伤方面的生物学行为,为临床应用提供物质基础。方法 含MyoD cDNA的质粒PEMMBC2 β5于大肠杆菌E.Coli DH5a内扩增;提取及纯化PEMMBC2 β5质粒;经DNA序列分析含MyoD cDNA;限制性酶切MyoD cDNA片段,连接酶连接到真核表达质粒pcDNA3-neo内,克隆出真核表达载体pcDNA3/MyoD。结果 提取及纯化的PEMMBC2 β5质粒含有大小正确的MyoD cDNA碱基片段,其碱基序列为编码目的基因的正确序列,凝胶电泳结果证明已将此片正确地克隆到pcDNA3/MyoD内。结论 成功的构建了MyoD基因的真核表达质粒pcDNA3/MyoD。     

人FasL全长cDNA的克隆及其真核表达载体的构建

目的：克隆人FasL全长cDNA并构建其真核表达载体。方法：采用RT-PCR法从健康人外周血单核细胞中扩增包含全部阅读框架的FasI。全长cDNA，将之与pGEM-T：Easy质粒连接、测序。构建pcDNA3．1-FasL真核表达载体，用脂质体介导方法转染人直肠癌8348细胞，检测FasL表达情况。结果：RT-PCR扩增的FasL产物经限制性内切酶酶切后生成的片段符合预期大小。克隆测序证实所得的FasL cDNA序列与Gene Bank序列完全一致。pcDNA3．1-FasL重组质粒经：EcoRI XhoI双酶切后，电泳显示898bp目的片段和pcDNA3．1载体片段，证明重组质粒正确。转染直肠癌8348细胞后。用RT-PCR方法检测FasL表达阳性。结论：采用RT-PCR方法成功克隆了人FasL全长cDNA并构建了其真核表达载体．为进一步研究FasL功能奠定了基础。     

Enzymatic action of prostate-specific antigen (PSA or hK3): substrate specificity and regulation by Zn(2+), a tight-binding inhibitor

BACKGROUND: In semen, prostate-specific antigen (PSA or hK3) digests the gel proteins semenogelin I and II, resulting in liquefaction and the release of motile spermatozoa. We characterized the substrate specificity and zinc-mediated inhibition of PSA. METHODS: The proteolysis of human semenogelin I (SgI) and II (SgII) by PSA was characterized by purification of generated SgI and SgII fragments, N-terminal sequencing, and mass spectrometry. Zn(2+)-inhibition of PSA was studied using a chromogenic substrate. RESULTS: Eighteen cleavage sites in SgI and 16 in SgII were identified. Cleavages were identified mainly as the C-terminal of certain tyrosine and glutamine residues, but also the C-terminal of histidine, aspartic acid, leucine, serine, and asparagine residues. No cleavages were identified at any arginine, lysine, phenylalanine, tryptophan, or methionine residues, indicating that the substrate specificity of PSA is distinct from that of trypsin, chymotrypsin, tissue kallkrein (hK1), and kallikrein 2 (hK2). Zn(2+) ions have a dramatic effect on PSA activity; the data indicate that Zn(2+) is a tight-binding inhibitor of PSA activity. CONCLUSIONS: The data will enable the optimized design of PSA activity assays, which may prove instrumental to uncovering the role of PSA in cancer and reproduction. The inhibition data indicate that Zn(2+) could regulate PSA activity, which may prove important in the development of efficient inhibitors of PSA activity.     

附睾蛋白酶抑制蛋白的研究进展

附睾蛋白酶抑制蛋白(Eppin)基因是最近克隆到的一种人类和小鼠附睾和睾丸中特异性表达的基因。Epp in蛋白是一种富含半胱氨酸同时含有乳清酸蛋白(WAP)和牛胰蛋白酶抑制蛋白(BPTI)结构域的分泌蛋白,它与精子成熟和生殖有关,此外还参与了人类附睾的天然免疫系统。针对Epp in蛋白的免疫避孕是一种有效和安全的方式,但还需进一步验证它的安全性。本文就Epp in的生殖和免疫功能及在免疫避孕中的应用进行综述。     

大鼠ETFβ基因过表达对NADPH氧化酶基因表达的影响

目的:构建大鼠ETFβ的重组质粒并检测其在人胚胎肾293T细胞中的表达和对NADPH氧化酶的影响。方法:应用RT-PCR方法从大鼠肾脏组织总RNA中扩增出编码ETFβ的cDNA,克隆至pUM-T载体并测序,然后亚克隆至真核表达载体pcDNA3.1/V5-His,酶切鉴定后测序,测序正确后用磷酸钙共沉淀法瞬时转染HEK293T细胞,使用RT-PCR方法检测空载体组和重组质粒转染组的NADPH氧化酶各亚基mRNA水平的表达变化。结果:(1)测序结果证实PCR扩增得到编码ETFβ的cDNA序列正确;(2)磷酸钙共沉淀法转染HEK293T细胞后,ETFβ融合蛋白成功表达;(3)RT-PCR结果显示重组质粒转染组的NADPH氧化酶5个亚基中的NOX3,NOX4的mRNA表达降低(P<0.05),空载体组和重组质粒转染组之间比较NOX1,NOX2,NOX5的mRNA表达差异无统计学意义。结论:成功构建大鼠ETFβ的重组质粒,该重组质粒可在HEK293T中过表达,并且降低了NADPH氧化酶NOX3,NOX4的mRNA表达水平。     

Expression, purification, and characterization of active recombinant prostate-specific antigen in Pichia pastoris (yeast)

BACKGROUND: Prostate-specific antigen (PSA), a member of the kallikrein family of serine proteases, is a chymotrypsin-like glycoprotein produced by the prostate epithelium. Elevated serum PSA (> 4 ng/ml) is a tumor marker for prostatic cancer and benign prostatic hypertrophy; increasing serum PSA over time is indicative of metastatic disease. It has been suggested that PSA may contribute to tumor metastasis through degradation of extracellular matrix glycoproteins, as well as cleavage of IGF binding protein-3, a modulator of IGF-1. To elucidate the role of PSA in the development and progression of prostatic cancer, it is necessary to have a reliable, cost-effective source of enzymatically active protein. Previous efforts to express recombinant PSA (rPSA) produced inactive proPSA, or mixtures of active and inactive PSA requiring activation by removal of the propeptide. We describe the expression of active recombinant mature PSA in yeast. METHODS: Stable chromosomal integration of a construct consisting of the yeast alpha-factor signal sequence preceding the mature PSA sequence resulted in secretion of rPSA. The rPSA was purified from the yeast cell culture supernatant to homogeneity by strong cation-exchange chromatography, and characterized by SDS-PAGE, Western analysis, electrospray mass spectrometry, N-glycanase digestion, N-terminal amino acid sequencing, and inactivation by a PSA-specific inhibitor. RESULTS: We report the production of active, mature rPSA in Pichia pastoris. Two forms of rPSA varying slightly in glycosylation were identified. The specific activity of the rPSA was equal to that of human seminal plasma PSA (0.56 micromol/min mg) as determined using a chromogenic substrate. CONCLUSIONS: Large-scale production of active rPSA will be useful in the exploration of PSA effects on tumor cell proliferation, migration and metastasis. In addition, a large supply of enzyme should facilitate the discovery of novel inhibitors for in vitro and in vivo evaluation, and may provide a reproducible source of rPSA for use as a standard in diagnostic testing.     

Antimicrobial peptides/proteins--application to the therapy of sepsis

Many antimicrobial peptides and proteins were discovered recently in various animals. Cecropins are insect-derived antimicrobial peptides which contain 35-39 amino acid residues. Magainins are amphibian-derived antimicrobial peptides with 21-27 amino acid residues. In mammals, defensins, 29-35 amino acid peptides, were identified in the granules of neutrophils and various epithelial cells. In addition, the granules of neutrophils in the mammal have been shown to have several antimicrobial proteins. Among them, bactericidal/permeability increasing protein (BPI) and cationic antimicrobial peptide-18 (CAP 18) have been found to have potent bactericidal activity against gram-negative bacteria and strong lipopolysaccharide-neutralizing function. The recombinant BPIs (recombinant BPI, 23-kDa N-terminal fragment of BPI, and lipopolysaccharide-binding protein-BPI fusion protein) and synthetic peptides derived from C-terminal of CAP 18 are now under investigation for the application to the therapy of sepsis or septic shock.     

中国人血管生成素-1cDNA克隆和序列分析

目的 克隆和测定国人血管生成素-1(Ang-1)基因序列。方法 巢式多聚酶链反应(PCR)从婴儿肺组织扩增中国人Ang-1cDNA，克隆入载体pGEM-Teasy，测定核苷酸序列，分析测序结果。结果 通过获得Ang-1cDNA，和GeneBank的Ang-1序列(U8508)对照，两者成熟区cDNA的核苷酸和氨基酸同源性分别大于99％和大于98％；在330bp～930bp问核苷酸同源性为98％，氨基酸同源性为95％。结论 获得克隆的中国人Ang-1基因成熟区。其卷曲卷曲结构域内可能存在一个内部高变区。     

Characterization of a 28-kDa collagenous protein extracted with EDTA from adult rabbit alveolar bone

Bone proteins in mandibular alveolar bone from young adult rabbits (3-month-old) were extracted with 4 M guanidine hydrochloride (GuHCl), followed by 0.5M ethylenediaminetetraacetic acid (EDTA). The proteins in the EDTA extract were fractionated on Sepharose CL-6B in the presence of 4 M GuHCl, followed by HPLC using hydroxyapatite and then ‘Mono Q’ resin in the presence of 7 M urea, and a 28 kDa protein was isolated. The purified 28-kDa protein showed intense staining with silver following SDS-PAGE separation under reducing conditions. When the protein was digested with bacterial collagenase, a 19-kDa fragment was produced. However, the protein was not susceptible to cyanogen bromide. On SDS-PAGE under non-reducing conditions, the protein migrated as two bands; a new band at about 85-kDa, and the original 28-kDa band. The amino acid composition of the protein was similar to that of α1-pN-propeptide of type I procollagen from other tissues. Moreover, the characteristics of the purified 28-kDa protein were similar to those of a 28-kDa protein synthesized by rabbit alveolar bone-derived cellsin vitro.