一种新的混合斑精子分离方法

doi:10.7503/cjcu20140965

摘要/Abstract

摘要：

制备了精子表面膜抗原受精素β蛋白(Fertilin β)的特异性抗体, 通过抗原-抗体反应和抗体固相偶联技术, 将抗体连接到琼脂糖球珠上, 建立了混合斑精子分离纯化的新方法. 首先, 通过聚合酶链式反应(PCR)扩增编码人受精素β蛋白第341~373位氨基酸的基因序列, 构建PGEX-4T-1/Fertilin β原核表达质粒; 其次, 诱导表达GST-Fertilin β融合蛋白, 制备受精素β蛋白多克隆抗体, 免疫荧光检测结果表明, 受精素β蛋白定位于精子的头后部, 并且阴道上皮细胞没有受精素β蛋白的表达; 最后, 将受精素β抗体与ProteinA琼脂糖球珠连接构建固相抗体系统, 将精子吸附于表面, 可从混合斑(精子与阴道上皮细胞混合液)中分离纯化精子. 本文为性犯罪案件侦破提供了新的方法.

关键词: 混合斑, 受精素β蛋白, 多克隆抗体

Abstract:

In sex crimes cases, mixed stain samples(a mixture of sperm and vaginal epithelial cells) are common and important biological evidence. Identification of sperm sources is the key of cases detected. The lack of effective technique of sperm separation from mixed stain samples is the bottleneck of sperm test. In the pre-sent study, the group chose sperm surface antigen, Fertilin β, as antigen to prepare specific antibodies, and further coupled the antibody to a solid phase of agarose beads, in order to establish a new sperm separation system. First, antigen Fertilin β recombinant plasmid was constructed. A cDNA for Fertilin β 341—373aa was amplified by polymerase chain reaction(PCR), and was cloned into a prokaryotic expression plasmid, pGEX-4T-1 vector. Second, GST-Fertilin β(341—373aa) fusion protein was induced and purified. Against this antigen, the Fertilin β antibodies were obtained from serum of rabbit immunized. ELISA and Western blot analysis demonstrated the specificity of the antibody against Fertilin β antigen. Then, the specific recognition of the antibody to sperm was identified by the immunofluorescence assay, in which Fertilin β protein in the sperm head can detected by the antibody, whereas in the vaginal epithelial cells showed negative detection. Finally, the sperm detection system using the solid phase of Fertilin β antibody was established, in which the Fertilin β antibody was coupled to ProteinA/G agarose beads and was able to separate sperm from the mixture. In conclusion, the Fertilin β antibody was successfully prepared, which was able to bind to the rear of the sperm head. Furthermore, the Fertilin β solid phase antibody system was generated, which could bind to the sperm surface, so as to isolate the sperms from the mixed stain samples. This sperm separation method from mixed stain samples provided a new detection for sexual offenses.

Key words: Mixed stain sample, Fertilin β, Polyclonal antibody

中图分类号:

O657

TrendMD:

伍贤军, 赵兵, 缪璇, 钱程, 王冰瑜, 李晓萌, 杨百全. 一种新的混合斑精子分离方法. 高等学校化学学报, 2015, 36(5): 886.

WU Xianjun, ZHAO Bing, MIAO Xuan, QIAN Cheng, WANG Bingyu, LI Xiaomeng, YANG Baiquan. New Method for Sperm Separation from Mixed Stain Samples^†. Chem. J. Chinese Universities, 2015, 36(5): 886.

图/表 10

Fig.1 PCR amplifying of 341—373aa from Fertilin βLane 1: DNA marker; lane 2: DNA band of 341—373aa rom Fertilin β.

Fig.2 Fertilin β recombinant plasmid sequencing resultDNA nucleotide sequence: GAATTCGAAGCAATTCATTTCAGTGGTGTGAAGATCTTTAGTAACTGCAGCTTCGAAGACTTTGCACATTT-TATTTCAAAGCAGAAGTCCCAGTGTCTTCACAATCTCGAG.

Fig.3 SDS-PAGE analysis of GST-Fertilin β fusion gene expression product in E.coliLane 1: Protein marker; lane 2: lysate of bacteria GST/BL21 before IPTG induction; lane 3: lysate of bacteria GST/BL21 after 0.1 mmol/L IPTG induction; lane 4: lysate of bacteria GST/BL21 after 0.5 mmol/L IPTG induction; lane 5: lysate of bacteria Fertilin β/BL21 before IPTG induction; lane 6: lysate of bacteria Fertilin β/BL21 after 0.1 mmol/L IPTG induction; lane 7: lysate of bacteria Fertilin β/BL21 after 0.5 mmol/L IPTG induction.

Fig.4 SDS-PAGE analysis of purified GST and GST-Fertilin β fusion proteinLane 1: Protein marker; lane 2: purified GST; lane 3: purified GST-Fertilin β.

Fig.5 Titer of Fertilin β immune serum etected by ELISA assay

Fig.6 SDS-PAGE analysis of ProteinA purified Fertilin β antibodyLane 1: Protein molecular weight marker; lane 2: unpurified Fertilin β antiserum; lane 3: proteinA purified Fertilin β antibody.

Fig.7 Western blot analysis of Fertilin β inmouse testisLane 1: Negative control protein sample; lane 2. lysate of mouse testis.

Fig.8 Immunofluorescence analysis of expression of Fertilin β in spermFertilin β was stained with Fertilin β antibody(red); nuclei were stained with DAPI(blue). (A), (E) DIPA; (B) PBS;(C), (G) DIPA with Fertilin β merge; (D), (H) PBS/Fertilin β with sperm merge; (F) Fertilin β.

Fig.9 Immunofluorescence analysis of expression of Fertilin β in sperm and vaginal epithelial cellsFertilin β was stained with Fertilin β antibody(red); nuclei were stained with DAPI(blue). (A) DIPA; (B) Fertilin β; (C) DIPA with Fertilin β merge; (D) fertilin β with sperm and vaginal epithelial cells merge(sperm, yellow arrow; vaginal epithelial cells, green arrow).

Fig.10 Images of ProteinA-Fertilin β antibody to capture sperm(A) ProteinA unable to capture sperm; (B)—(D) proteinA-Fertilin β antibody to capture sperm(sperm, solid arrow; ProteinA, hollow arrow).

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