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人类BSP蛋白相关新基因的cDNA克隆、定位和表达
【作者】 罗阳；
【导师】 于秉治；
【作者基本信息】 中国医科大学 ， 细胞生物学， 2002， 博士
【摘要】 前言 BSP（bovine seminal plasma）蛋白是牛精浆中的主要蛋白质，BSP蛋白可以与胆碱或磷脂胆碱结合，黏附于精子表面，可促进由肝素、HDL等诱导的获能过程，另外BSP蛋白与不同来源的胶原、纤维蛋白原、肝素、钙调蛋白、IGF-Ⅱ、apoA-Ⅰ等结合，BSP蛋白还可抑制精子胞膜上的胆固醇迁移，这些都暗示了BSP蛋白在精子胞膜的脂类代谢及调节精子的获能以及顶体反应方面发挥重要作用。目前在猪及马的精浆中都发现了与BSP蛋白同源的蛋白质的存在，这些蛋白具有与BSP相似的结合特性，说明BSP蛋白及其同源蛋白在哺乳动物中是广泛存在的，并具有其生理意义。而在人的生殖系统中与BSP功能相关的蛋白质至今未见报道。为了进一步发现和研究BSP及其相关蛋白在受精及受精卵发育中的重要作用，我们克隆出人与BSP蛋白相关基因的cDNA，将其命名为HBRP（Human BSP-Related Protein）。经BLAST分析，在GenBank未检索到与之相同的蛋白，我们对HBRP基因的染色体定位以及在人体各组织中的表达分布情况进行了研究，并将该基因编码的蛋白进行了原核表达。通过基因重组技术大量获得表达蛋白，并检测了其对蛋白激酶C（PKC）活性的影响，对进一步研究新蛋白的生物学功能具有重要的意义。 材料与方法 1．3′-RACE和5′-RACE PCR反应 参照GenBank数据库人睾丸组织表达序列标签（expressed sequence tag，EST）序列（AA429491），设计3′-RACE（Rapid Amplification of cDNA Ends）基因特异性引物，以Human Testis Marathon-Ready cDNA为模板，使用MarathonTMcDNA Amplification Kit和Advantage DNA Polymerase，采用巢式PCR（Nested PCR）方式，进行3′-RACE PCR反应。在此基础上设计5′-RACE PCR反应引物，进行5′-RACE PCR反应。1％琼脂糖凝胶电泳检测扩增产物。 2．山NA的克隆和测序 将得到的 3’一及 5’－sCE PCR产物符合预期长度的片段，分离后纯化、回收，克隆到TA克隆载体PMD－18T，转化E．COlt JM109感受态细胞，菌落PCR筛选鉴定阳性克隆，用M13通用引物进行DNA序列测定。测序结果经微机处理拼接，确定正确CDNA序列。在序列的两端设计引物，PCR扩增全长CDNA，克隆后测序分析。 3．全长山NA序列及其编码蛋白质的计算机分析 从CDNA的序列推导出它所编码的氨基酸序列，应用NCBI提供的BLAST程序在 GenBank数据库和蛋白质数据库进行同源性比较分析，在N－tP：／／。exPasy．oh／csi－biVscanProsite分子生物学网站中进行蛋白质的功能基序分析。 4．HBRP基因的染色体定位 使用 Stanford G3 RHkadiation hybrid)嵌板，在所要定位基因的 3’非翻译区（untranslated region，UTR）设计引物，预先扩增人基因组DNA，电泳检测产物大小，继之扩增G3 RH panel，重复两次，将PCR结果输人斯坦福人类基因组中心（SHGC）的主页(http：／／、－ShgC．S格五b＊·edVRH／H／rhserverfo…ew．htlnl人得到与待定位基因距离最近的SHGC标签号，最后借助于RH图谱和基因组遗传图谱找到相对应的染色体区带。 5．RT—PCR 采用TriZOI一步法提取正常组织总RNA，反转录合成CDNA。于HBRP基因的第23外显子处（跨过一个内含子）分别设计上下游引物，引物之间的山NA长度为250hP，PCR检测HBRP基因在人体各组织中的表达分布。应用人p－actin引物进行RT－PCR作为内对照。1．5％琼脂糖凝胶电泳检测表达结果。 6．HBRP表达质粒的构建 以克隆在PMDJ8T的全长CDNA为模板进行PCR扩增，在引物序列5’端引人 BamH酶切位点和起始密码，3’端引人 Xho酶切位点，分别将PCR产物和 GST基因融合载体pGEX－SX一且用 BamH和 Xho双酶切，回收纯化后，16℃3 h完成连接反应，转化 E．COlt B＊ 感受态细胞，菌落PCR筛选鉴定重组阳性克隆，质粒提取，B。mH和 Xho双酶切消化鉴定 ·2·插人片段。选择阳性克隆做测序分析，确定正确阅读框架克隆。 7．融合蛋白的表达 将正确读框的单克隆菌以及含母本 PGEX质粒的转化菌在2 X YT培养基中(含AmPicillin 100 m牙L乃7丫：培养至A。为0．5－1时，加人终浓度为0．InunoUL的I工G，诱导表达3h，离心收集菌体，PBS洗涤，超声波破碎，加人 Tritoall一 至终浓度为 1％后混匀，离心收集上清，即为蛋白质粗提液，SDS－PAGE检测表达情况。 8．Western印迹分析 经初步纯化的融合蛋白用12％SDS－PAGE分离，然后转移到硝酸纤维素膜上，用含二％BSA的 TBST封闭。抗 GST抗体用作一抗，碱性磷酸酶偶联的羊抗鼠IgG作为二抗，用N13T／BCIP底物显色。 9．重组蛋白对PKC活性的影响 从大鼠脑中分离纯化蛋白激酶 Chrotein kinase C，PKC厂LJ Histone H；为底物，＊／zP」－ATP标记测定 PKC活性，收集有活性部分，采用考马斯亮兰法测定蛋白浓度。使用 Glut。Lthione SePharose 4B纯化表达的 GST和GST－HB即融合蛋白，分别检测不同浓度的GST蛋白及GSP－HB地融合蛋白对PKC活性的?
【Abstract】 BSP ( boyine seminal plasma) protein is the cardinal protein in bovine sperm, which can combine with choline or phosphycholine and then adhere to the surface of sperm to promote the sperm capacitation induced by heparin and high density lipoproteins ( HDL ). Moreover, BSP protein can combine with heterogeneous collagen, fibrinogen, heparin, calmoduline, IGF - II and apoA - I . BSP can also inhibit the migration of cholesterol on the sperm membrane. All these indicate that BSP protein plays an important role in the lipid metabolism of sperm membrane and the modulation of sperm capacitation. At present, homogeneous proteins of BSP proteins found in the sperm plasma of horse and pig have the binding - properties similar to BSP protein, which indicates that BSP protein and its homogeneous protein exist commonly in mammals and show their physiological functions. However, in human congenital system proteins associated with BSP in function have not been reported up to date. To find BSP proteins and its related proteins important roles in fertilization and the development of the zygote, we clone the full - length cDNA which is related to BSP genes and named HBRP( Human BSP - Related Proteins). We can not find the same protein in Genbank through the BLAST procedure from NCBI. We located HBRP gene on the chromosome and examined the expression of HBRP gene in human tissues. We also expressed HBRP in E. Coli. We have obtained amount of expression proteins through the gene recombinant technique and detected its effect on the activity of PKC, which is crucial to further study.Materials and methods1. 3’-RACE and 5’-RACE PCR reactionTwo specific 3’- RACE primers are designed by the reference to the ESTcDNA sequence ( AA429491) from GenBank date House of human testis tissue. Using Human Testis Marathon - Ready cDNA as template, the 3’- RACE PCR reaction is performed by using the Marathon?cDNA Amplification Kit and Advantage cDNA Polymerase. On the basis of above work, 5’- RACE PCR reaction primer is designed. 5’- RACE PCR reaction is followed and analyzed in 1% agarose/EtBr gel.2. Cloning and analyzing of the full -length cDNA sequenceThe fragments consistent with expected length from the product of RACE -PCR reaction are separated, purified and recovered, after which the fragments are cloned into the vector of pMD - 1ST to transform the competent cells. The positive clone is selected by colony PCR and DNA sequence is analyzed and determined by the M13 primer. Then the correct sequence is determined by the computer processing. The full - length cDNA of HBRP is amplified by PCR reaction and sequenced after cloning.3. Computer analysis of the cDNA and its encoding proteinThe amino acid sequence encoded by full - length cDNA is deduced by u-sing the BLAST procedure from NCBI. The homogeneous comparison and analysis are performed in the GenBank and protein Date House, while the functional residual sequence of the protein is analyzed in the molecular Biological net, Ht-tp://www. expasy. ch/cgi - bin/scanpaosite4 . Location of the HBRP gene in the chromosomeAccording to Stanford G3 RH panel, a pair of primers are designed in the 3’ - UTR region of the gene which is to be localized and human genome DNA is amplified in advance whose products are examined by electrophoresis. Then G3 panel is amplified and this process is repeated twice. The PCR result is sent to Stanford Human Genome Center (SHGC) http://www - shgc. Stanford, edu/ RH/rhserverformnew. html, obtaining the nearest label number of SHGC to the gene which is to be localized, eventually, finding the corresponding chromosome band through the RH graphics ahd genome hereditary graphics.5. RT-PCRThe total RNA of normal tissues is extracted by using the method of TrizolReagent. The first chain of cDNA is synthesized through reverse transcriptase. The primers are designed in sites of the number 2 and 3 exon of HBRP gene. The product of RT - PCR by using human B - actin as primer is looked on as the positive control. The result i