【作者】 王东辉；

【导师】 柳忠辉；

【作者基本信息】 吉林大学 ， 免疫学， 2004， 硕士

【摘要】 Follistatin (FS，卵泡抑素 ) 在机体内主要以二种形式存在，即288氨基酸残基组成的FS-288形式及315氨基酸残基组成的FS-315形式，具有抑制垂体前叶腺细胞分泌促性腺激素(FSH)、特异性结合激活素等作用。为了探讨FS在相关疾病诊断及治疗方面的作用，本研究制备了抗FS315 C末端多克隆抗体，并在本室已制备的抗FS N末端单克隆抗体基础上，建立了FS315双位点ELISA检测法，并且对正常及患者血清中FS315水平进行了检测。具体研究内容如下。1．重组人FS-315 C末端肽融合蛋白原核表达载体构建（1）钓取编码人FS-315C末端肽的cDNA：提取人新鲜卵巢组织总RNA，用Takara一步法试剂盒扩增FS-315C末端cDNA，采用DE81纤维素法回收特异性PCR产物。（2）TA克隆：将PCR扩增的FS-315C目的基因片段与pGEM-T质粒进行TA克隆连接，取连接产物转化感受态大肠杆菌JM109，采用蓝白筛选的方法筛选阳性克隆。（3）TA克隆阳性重组质粒的鉴定：碱裂解法提取质粒，鉴定含有目的基因片断的阳性质粒，Applied Biosystems 100 Model 377型DNA序列自动分析仪测定目的基因序列，所克隆的FS-315C末端基因核苷酸顺序与Gene Bank所载一致，命名质粒为pGEM-FS-315C。（4）原核表达质粒pGEX-FS-315C的构建及鉴定：通过EcoRⅠ和XholⅠ双酶切，将pGEM-FS-315C中目的基因片段定向插入GST融合蛋白表达载体pGEX4T-1中。电转化感受态大肠杆菌DH5α，质粒抽提及酶切鉴定，结果与预计一致，再经5’端上游引物测序，选取序列正确的pGEX-FS-315C克隆。2．GST-FS融合蛋白的诱导表达及产物的SDS-PAGE鉴定：（1）GST-FS融合蛋白的诱导表达及鉴定：表达质粒pGEM-FS-315C <WP=47>电转化E.coli BL21，IPTG诱导蛋白表达，超声波破碎菌体，离心后取上清，上清含有分泌型GST-FS融合蛋白。用10%SDS-PAGE鉴定融合蛋白表达情况，GST-FS融合蛋白表达量约占菌体总蛋白的20-30%。（2）GST-FS融合蛋白的纯化：Glutathione-Sepharose 4B亲和层析柱纯化表达的GST-FS-315C融合蛋白，按试剂盒说明书进行，纯化的GST-FS-315C融合蛋白再经电泳纯化，最终获得重组GST-FS-315C。FS315双位点ELISA检测法的建立（1）抗人FS-315C末端多克隆抗体的制备：用纯化的GST-FS315C免疫家兔，获得抗FS抗血清，抗血清经硫胺沉淀、ProteinA亲和层析纯化，获得抗FSC末端多克隆抗体。( 2 ) 双位点酶联免疫吸附试验（ELISA）检测人FS315：采用抗人FS N末端单克隆抗体（本研究室制备）包被酶标板，抗人FS-315C末端多克隆抗体为检测抗体，建立FS315双位点ELISA检测法；并利用该法分析了多种生物样本中的免疫反应性FS（Ir-FS），其结果在正常人血清、卵泡液、羊水以及小细胞肺癌培养上清中均检测到了FS315。实验同时采用IRMA检测法对上述多种样本中的FS水平进行了分析，采用ELISA和IRMA检测血清和羊水中FS水平，结果显示二者检测结果高度一致（r=0.90和0.92）；卵泡液和培养上清中的FS水平采用ELISA和IRMA检测的结果也极其相近，二者检测结果高度一致（r=0.92和0.95）。上述资料表明，本研究建立的FS315双位点ELISA检测法，适用于多种样本中的FS315水平测定。该ELISA检测样本中follistatin灵敏度为0.25ng/ml，未见与rh-Activin、Inhibin、ACTH、CRF、FSH和LH有交叉反应。实验结果表明所建立的双位点ELISA法是一种简便、快速、稳定、高敏感度及特异性的follistatin检测系统，可用于血清、羊水及卵泡液等多种生物样本中的FS检测。4．成年人外周血FS水平的随龄变化正常体检的健康成年人按年龄分组，ELISA法检测血中FS水平，同 <WP=48>时采用放射免疫分析方法(RIA)测定血中激活素A水平变化。结果显示健康成年人血清FS315水平随年龄呈正相关增加趋势，与激活素A的变化一致。5．孕期女性外周血FS水平变化采用建立的ELISA方法测定了64例正常妊娠女性血中FS水平变化情况，同时采用放射免疫分析方法测定了这些孕期女性血中的ActivinA水平变化。结果显示妇女怀孕期血清中follistatin及ActivinA水平随孕龄增加而增加。6．ELISA法检测部分患者血清中FS水平应用ELISA法检测部分患者血中follistatin水平，结果显示如慢性肝肾疾病以及血液的恶性肿瘤患者外周血FS水平升高。上述资料显示本次建立的FS315双位点ELISA法适合多种生物样本中的FS315水平测定，研究结果显示FS315及激活素A随年龄呈正相关变化，在孕期女性血中随孕龄增加而增高，可能与胎儿的发育成熟有关。该研究还显示FS315水平变化可能与机体疾病有关，如在慢性肝肾疾病时FS水平升高。更多还原

【Abstract】 There are two major species of follistatin (FS) in vivo, which are FS-288 consisting of 288 amino acid residues and FS-315 consisting of 315 amino acid residues and play important roles in inhibiting the secretion of FSH from anterior pituitary cells and specifically binding with activin. In this study, in order to investigate the effects of FS in the related diseases, we established two-site ELISA for detecting the FS-315 level in the serum of normal people and patients by using anti- FS-315 C-terminal polyclonal antibody and anti-FS N-terminal monoclonal antibody. The detail procedures are as follows. 1. Construction of FS expression vector of C-terminal peptides of human FS-315 (1) Preparation of cDNA encoding C-terminal peptides of human FS-315 protein: we extracted the total RNA from fresh human ovary cells and amplified the cDNA for C-terminal peptides of FS-315 by using Takara one-step RT-PCR kit, and recovered specific PCR products by electrophoresis onto a sliver of DE81 cellulose membrane.(2) TA cloning: we ligated the target gene fragment of FS-315C and the pGEM–T plasmid by TA cloning, after that we transformed the recombinant plasmid into the JM109 competent cells. We obtained the positive clone by blue-white color test.(3) Identification of the positive recombinant plasmid: we extracted the positive plasmid by means of alkaline lysis with SDS, identified the positive plasmid which contained target gene fragment, sequenced by the Applied Biosystems 100 Model 377 DNA sequence auto-analyser, and the sequence of FS-315C is identical with GeneBank, so the plasmid was named as pGEM-FS-315C. (4) Construction and identification of prokaryotic expression plasmid of <WP=50>pGEX-FS-315C: We inserted the target gene of pGEM-FS-315C into pGEX4T-1, a GST fusion protein expression vector, after using EcoRⅠand XholⅠto digest the plasmid, we transformated it into E.coli DH5αby electroporation and extracted the plasmid and identificated it by restriction enzyme digestion, the result was identical with that we had expected. After sequencing with the 5’—primer, we choosed the clone of pGEX-FS-315C with correct sequences .2. IPTG-induced expression of GST-FS-315C fusion protein and identification of the production by SDS-PAGE:(1) IPTG-induced expression and identification of GST-FS-315C fusion protein: expression plasmid for pGEM-FS-315C was transformed into E.coli BL21, induced the expression of protein with IPTG , and brake the bacteria by sonication, after centrifugation, collected the supernatant containing the secretive GST-FS fusion protein, At last we used 10% SDS-PAGE gel to identify the expression of the fusion protein, the expressive GST-FS fusion protein accounts for about 20% of the total bacterial proteins.(2) Purification of GST-FS-315C fusion protein: we purified the GST-FS fusion protein by Glutathione-Sepharose 4B affinity chromatography column according to its instruction, and the purified GST-FS fusion protein was purified by SDS-PAGE, then we obtained the recombinant GST-FS-315C.3. Establishment of the two-site ELISA for FS-315(1) Preparation of the anti-human FS-315C terminal polyclonal antibody: we immunized the New Zealand rabbits with the purified GST-FS-315C, and obtained the antiserum of FS, then precipitated the antiserum with saturation ammonium sulfate and then purified it by ProteinA affinity chromatography. Thus we got the polyclonal antibody of the C-terminal peptide of FS.(2) Measurement of human FS-315 by Two-site ELISA : we used the anti- human FS N-terminal monoclonal antibody to coat the 96-well plate, and the <WP=51>anti-FS-315 C terminal polyconal antibody as the testing antibody. After that we established two-site ELISA analysis. By using this method we analyzed immunoreactive FS (Ir-FS) in many biological materials, and found FS-315 in serum , follicular fluid, amniotic fluid of normal people and the supernatant of cultured small cell lung cancer .In this study we also used IRMA to analyze the level of FS in many samples mentioned above. We use更多还原