人精浆蛋白双向谱型特征及其器官特异性的研究

本文应用双向聚丙烯酰胺凝胶电泳（2D—PAGE）技术分析了60例正常人精浆中的蛋白成分。阐明了人精浆蛋白双向电泳谱型的特征，并将人精浆蛋白双向电泳谱型与其他体液蛋白双向谱型进行了比较，证明人精浆蛋白双向电泳谱型具有器官特异性。本文还用2D－PAGE对不同条件下保存的精斑进行了认定。并将其应用于强奸案的鉴定。     

ELISA检测精浆特异蛋白P_(30)鉴定人精斑

<正> 1978年美国Sensabaugh分离出精浆特异蛋白P30,并成功的制备出相应的抗P30血清。1987年我国也研制出抗人精浆特异蛋白P30血清,并应用该种抗血清建立了几种琼脂扩散和电泳方法鉴定人精斑。由于人精液中P30含量不高,平均为1.52±0.676mg/ml,鉴定微量精斑存在困难。为了更有效的提高方法的灵敏度。我们建立了鉴定微量人精斑的ELISA固相P30抗体法,现报告如下。     

抗人精浆特异蛋白血清的制备与纯化

本文报导检验混合斑中精斑ABO 血型所用抗人精浆特异蛋白(anti-human seminal peculiarprotein,ASPP)血清的制备及用溴化氢活化琼脂糖4B 为载体的亲和层析纯化抗血清方法,并指出纯化时的最佳条件。该血清可准确地分离混合斑中精斑的ABO 血型。     

用酶免疫斑点法快速检测精浆特异蛋白P_(30)鉴定人精斑

作者用自制的辣根过氧化物酶标记的抗精浆特异蛋白P_(30) 单克隆抗体,建立了简便快速检测精斑中P_(30) 鉴定人精斑的斑点法。通过颜色变化判定结果,阳性为紫蓝色斑点,阴性为无色。结果表明,仅人类精斑和前列腺浸液出现阳性斑点,人体其他体液及组织器官浸液均为阴性。对动物血、精斑也无交叉反应。标定精斑稀释到1600倍亦可得到阳性结果。     

抗人精液血清特异性的研究

确证精斑常用抗人精液血清作沉淀反应。这种抗血清特异性差,常与人类某些体液发生交叉反应,以致精斑确证试验结果不可靠,影响鉴定结果的准确性。作者用人类精子、精浆、精液与人类精浆特异性抗原p30分别免疫家兔,制备各种抗血清,用环状沉淀、琼脂双向扩散及免疫电泳等技术对这些抗血清的特异性进行了研究,报道如下。     

正常月经周期中阴道液蛋白SDS-PAGGE谱型及特异性

采用 SDS-PAGGE,研究了由21个个体提供的共25份(其中4人提供两个周期)月经周期的阴道液蛋白电泳谱型。结果发现阴道液电泳谱型变化与月经周期无关。通过与其它体液对比发现,阴道液电泳具有特异蛋白带,有器官特异性。     

人发毛干角蛋白电泳谱型的分析

本文用SDS-梯度(5.0～17.0％)聚丙烯酰胺凝胶电泳对150例中国人头发角蛋白组分进行了分析。结果表明,在150例人头发中,有6种不同类型的角蛋白电泳谱型。此1～6型发生频率分别为24％、12％、42％、7.3％、8.0％;及6.7％。用N-(3-芘)马来酰胺标记头发角蛋白巯基,证实人头发不同类型的角蛋白电泳谱型主要区别在低硫蛋白部位。作者认为,人头发角蛋白电泳谱型的差异可为法医学鉴定中的毛发个人识别提供重要的依据。     

用胶乳凝集抑制试验鉴定人类精斑

作者用人类精浆致敏的胶乳和人初乳吸收后的抗人精液血清,采用胶乳凝集抑制试验凹玻板法盲测了180份生物性斑痕,结果表明此法确证人类精斑的敏感性,准确性均高于精子检出法,而与抗 p30血清琼脂双向扩散结果一致,且可用于混合斑的检验,具有快速、准确、简便等优点。     

GC/MS衍生化测定精浆中游离支链氨基酸和果糖

目的 建立人精浆中支链氨基酸和果糖的气相质谱衍生化分析方法.方法 精液样本液化离心后,按精子活率的临床标准分为正常组和非正常组,经TMS微波衍生化,GC/MS分析两组支链氨基酸、果糖衍生物含量差异.结果 正常组与非正常组的缬氨酸,亮氨酸,异亮氨酸在相对含量上均有一定差异.正常组3种氨基酸相对含量高于非正常组.果糖分析观察到同样的特征,正常组相对含量高于非正常组.结论 精子活率低的精浆样本仍可检出支链氨基酸和果糖,其差异有助于无精子、少精子精斑的法医学鉴别.     

Chelex快速处理法成功检测肛门拭子1例

在实际办案中 ,法医检验精斑多集中于阴棉、内裤、擦拭物 ,而肛门拭子检验较少 ,笔者遇到一例 ,并成功检出犯罪嫌疑人所留精斑的基因型。2 0 0 2年 3月 ,谢某 (女 ,4 2岁 )在中山市东区某出租屋被杀。提取死者谢某阴棉、肛门拭子及嫌疑人曾某、于某、周某等人血纱 ,进行ＰＣＲ检验比对。血痕、混合斑ＤＮＡ提取见文献[1] 。ＤＮＡ扩增 :采用ＰＥ公司Ｐｒｏｆｉｌｅｒｐｌｕｓ试剂盒扩增。电泳采用 310遗传分析议电泳分型。第一次检验事主阴棉中精斑 9个ＳＴＲ位点基因型均与嫌疑人曾某相同 ,偶合概率为 1.3× 10 -12 ,从而认定精斑为曾某所…     

人精浆特异蛋白P_30的分离纯化与鉴定

本文介绍人精浆特异蛋白P_(30)分离纯化及鉴定的方法。人精浆经蒸馏水透析后经Sephadex G100、G150和G75三种不同的柱层析即可获得精浆特异蛋白。SDS-PAGE、免疫电泳、免疫双扩散证明其纯度和特异性均符合要求。它与已知抗P_(30)血清呈强阳性反应,用其免疫制备的抗血清和已知抗P_(30)血清,在免疫电泳中只与精浆或精浆特异蛋白形成一条完全相同的沉淀线,SDS-PAGE测得分子量约为30,000。故笔者将其亦定名为P_(30)。     

Polymorphism of seminal gamma-glutamyl transpeptidase

Electrophoretic analysis of seminal gamma-glutamyl transpeptidase (GGT) activity of 147 unrelated Japanese males revealed three types of band patterns. An anodal single band, a cathodal single band and heterozygous double bands termed 1, 2 and 2-1, respectively, were commonly identified in the samples. The frequencies of the three types were 1 = 0.22, 2 = 0.33 and 2-1 = 0.44. Seminal stains kept for more than 6 months revealed distinguishable band patterns as well as fresh samples.     

制备抗人精液特异蛋白P30血清的研究

作者以精浆特异蛋白P30为抗原免疫新西兰白兔、豚鼠和鸡三种实验动物,制备了抗P30血清。用双向琼脂扩散法检测兔和豚鼠的抗 P30血清,其特异性和敏感性均达到目前国外同类产品的水平。抗 P30血清与阴道分泌物,血清、唾液、尿液、初乳以及羊精液、鸡精液均不出现交叉反应。用抗 P30血清检测混合的人精浆,其抗原效价为1:160;P30含量可测到12.5ug/ml。在三种动物的抗血清中,豚鼠抗 P30血清的抗体效价最高。以不同浓度的 P30测豚鼠、兔和鸡抗 P30血清抗体效价豚鼠平均滴度可达52.50,兔次之,鸡的抗 P30血清最差。经作者制备的抗 P30血清可用来确证精液。     

人类精子ABO血型抗原的间接免疫荧光研究

应用间接免疫荧光技术,对40份精子标本进行 ABO 血型抗原检测。A 型人精子上存在 A 抗原;B 型人精子上存在 B 抗原;AB 型人精液中,一部分精子带有 A 抗原,另一部分精子带 B 抗原。各血型人的精子均有 H 抗原。精子血型抗原为本身所固有,并非来源于精浆。不同人的精子血型抗原含量各不相等,与其供体是否为分泌状态或强弱无直接关系。精子 ABO 血型抗原主要存在于精子的颈部和顶体等区域。     

混合斑中精斑ABO血型的检验

<正> 1988年,壹岐裕志等报告了用吸附抗α_2-SGP 血清的硝化纤维素膜(NCF)检验混合斑中的精斑 ABO 血型,但耗时。本文作者通过对此方法的改进,采用常彩琴等研制的抗人精特异蛋白血清(anti-human seminalpeculiar protein,ASPP),采用蛋清粘片热解离法检验混合斑中精斑 ABO 血型,耗时短,效果好。现介绍如下。     

Application of an immunological method to detect trace amounts of dried human semen

An immunological assay based on a monoclonal antibody was used for identification of trace amounts of dried human semen in forensic science evidence. The monoclonal antibody (Mab 4E6) produced recognizes a human sperm-coating antigen which is specific to human seminal plasma. This antigen seems to be a protein secreted by the epithelial cells of the ejaculatory duct, which is stable indefinitely at room temperature. Mab 4E6 reacts positively with semen samples from individuals independently to their ABO group or secretory status, but does not react with semen from bull, ram, boar, horse, rabbit and dog. In the assay system developed, Mab 4E6 can detect human seminal plasma at concentrations of 0.5 micrograms/ml total protein. A similar sensitivity is found when human semen stains are eluted from forensic science samples and tested by the same assay. This method shows a good correlation with the microscopic methods routinely used. The method described is very sensitive and reproducible, it is time saving and special laboratory equipment is not needed.     

Immunologic characterization of human seminal leucine aminopeptidase (LAP) and its medicolegal use

A new method for identification of seminal stains is described, based on the immunologic demonstration of leucine aminopeptidase (LAP), which is extremely abundant in human semen and specific for the prostate as well as semen. An antiserum against human seminal plasma was obtained by repeated immunization of rabbits with seminal plasma and Freund's adjuvant. Ouchterlony's double immunodiffusion test and Culliford's precipitin electrophoresis were performed to demonstrate specific proteins of seminal plasma. LAP activity was visualized with L-leucyl-beta-naphthylamide as substrate and with Fast Garnet GBC as coupler. The immunologic analysis of LAP produced two precipitin lines with enzyme activity. One was observed in kidney, jejunum, pancreas, prostate, as well as in semen, and was completely absorbed with kidney homogenates. The other was found only in semen and the prostate and was not absorbed with kidney homogenates. When the anti-seminal plasma serum absorbed with the kidney was used, the semen-specific LAP could be demonstrated by precipitin electrophoresis only in seminal stains stored for up to 2 months, whereas it was not demonstrated in stains from other human body fluids. By means of precipitin electrophoresis the detection of the semen-specific LAP was possible at semen dilutions of up to 1:32. The method described here greatly enhances the value of semen identification and is quite recommendable for the examination of stains in medico-legal practice.     

分泌抗人精液特异蛋白P_(30)单克隆抗体杂交瘤细胞系的建立和初步应用

作者通过杂交瘤技术建立了9株产生抗精浆特异蛋白 P_(30) 单克隆抗体的杂交瘤细胞系。它们是由 SP2/0骨髓瘤与经 P_(30) 免疫的 BALB/C 小鼠脾细胞按常规方法进行细胞融合、并经克隆化筛选得出.这些细胞株均经体外培养3个月以上能够稳定分泌抗 P_(30) 单克隆抗体。该抗体只能识别纯化的 P_(30) 和精液中的 P_(30) ;与人精液以外的其他体液和多种人体组织无交叉反应;与几种常见动物的精液和血液无交叉反应。这些 P_(30) 单克隆抗体均属 IgG 类和 IgG_1亚类。其培养上清液和腹水的抗体效价最高分别达到320和128,000。以 ELISA 法应用这些单克隆抗体能很好地鉴定精液和精斑。     

Identification of human semenogelin in membrane strip test as an alternative method for the detection of semen

Semenogelin (Sg), a protein originating in the seminal vesicles and a substrate for prostate specific antigen (PSA or p30), is a useful marker for the identification of semen. And detection of Sg has been available commercially in a membrane test recently. PSA is commonly used to detect semen in forensic significant samples taken from sexual assault cases. The strip PSA test has been available commercially from various manufacturers for many years. In this study, we evaluated two immunochromatographic membrane tests, one for Sg and the other for PSA by analyzing human semen, other human bodily fluids/materials including urine, blood, saliva, sweat, breast milk, vaginal secretion and fecal materials, semen from various animals and forensic casework samples. The data demonstrate that both Sg and PSA strip tests provide rapid and sensitive method for identification of seminal plasma. These results show that the immunochromatographic method for Sg detection is useful for the identification of seminal plasma in forensic samples, an alternative to the method for PSA detection.