抑制素A的克隆表达以及单克隆抗体的制备与鉴定

发布时间：2018-09-11 17:26

【摘要】：抑制素A是一种(32kDa)大分子糖蛋白肽类激素,由α及β两个亚基以二硫键(—S—S)偶联构成,属于转化生长因子β(TGF2β)多肽家族。其基本作用是抑制垂体产生卵泡刺激素(FSH),卵泡抑制素也在性腺中通过自分泌和旁分泌的形式调节卵泡细胞的分化和甾体的生成;大多数循环抑制素由性腺产生,另外它们在许多性腺外的组织,包扩脑,肾上腺,骨髓,胎盘都有表达并且在局部都有一定的调节作用。抑制素A不仅与卵泡发育和卵巢肿瘤有关而且近年来还发现它可以作为孕中期唐氏综合症筛查的一项指标。唐氏综合症(Down's)是人类最早发现且最常见的常染色体病,也是造成儿童先天性智力低下的重要原因之一。唐氏无明显的家族史,每个孕妇都有生出该患儿的可能性,至今尚无有效的治疗手段,所以产前诊断就显得较为重要,目前开展的羊水穿刺及绒毛活检仅限于高危人群,仅能检出20%的唐氏患儿,此外羊水穿刺及绒毛活检均为侵入性检查,不适于群体检查,为了能最大限度减少产前有创检查降低唐氏患儿的的出生率,产前的血清学的筛查已被广泛用于临床。现在妊娠中期的血清学检测主要采取AFP+HCG+UE3的三联实验,但是这些标志物的血清水平在正常和非正常人群都有所重叠,所以,新的标志物的发现和应用综合的检测方案是提高检出率的有效的方法。自从发现抑制素A的血清水平在怀有唐氏患儿的孕妇的血清中的水平高于正常妊娠后,抑制素A已被作为检测唐氏综合症的一个有效的标志物。 Down's综合征妊娠中母血标记物浓度变化机制尚不清楚,但胎儿产物或胎儿胎盘产物(AFP,uE3)减少,而胎盘产物(HCG,DIA)增加,证实了母体血浆中抑制素A等成分主要来自胎盘而不是胎儿。抑制素A作为Down's综合征孕中期血清筛查的第4个标记物,在妊娠时,抑制素A主要由胎盘分泌并且有活性的二聚体的形式(DIA)在血液中的水平明显升高。妊娠前三个月中DIA的血清水平升高随后大约在第10周下降,在第15-25周期间产妇的血清中的水平保持一个稳定的水平一直持续到妊娠末期达到峰值。它的这种稳定分泌周期使DIA的检测更加优化于其它的一些产前诊断指标。自从检测到在怀有唐氏综合症的产妇血清中抑制素A的水平高于正常孕妇的两倍或更高,DIA已经和其他产前诊断的标志物都作了比较和对照,发现在妊娠的中期联合AFP+HCG+UE3,可以将检出率提高八个百分点达到84%,并且假阳性率只有5%。另外HCG在妊娠15-20周含量变化较大而相对的DIA的水平却相对稳定,所以有许多学者认为DIA的检测可能更优于HCG。我们都知道妊龄多是根据末次月经所得。但是这样所得的结果可能存在误差,和其他血清标志物相比较,DIA的水平在妊娠的第二和第三个月不会随着妊龄的变化而有很大的波动,这在研究更可靠的数据去评价唐氏综合症时,它有很大的优势去解释这些结果,并且不会被不正确的妊龄所影响。随着现在人们对唐氏综合症的危害的了解,大家都希望能在没有危险的情况下进行早期诊断,早期终止妊娠。所以,又有实验室用DIA联合NT,PAPP-A,β—HCG进行了筛查,当结合年龄若加上DIA则可以使早期诊断率提高到90%。早期抑制素A的测定在检测DIA和α亚单位的游离形式之间存在一定困难,因此在检测结果中出现了许多不确定的值,但是随着针对DIA的特异性抗体的出现使得DIA的检测不在是难事。现在唯一能测定抑制素A皮克水平的方法仍是免疫测定法。这种方法都已被证实可以用来特异性的检测人类血浆中DIA的水平,这种方法都是双夹心酶联免疫吸附实验随着这些检测技术的发展,使得各个实验室的筛查结果也有了很大的差异,有报道说在比较怀有唐氏的孕妇和正常的孕妇抑制素A的水平时发现在7-8周时其水平降低而在9-11周时升高。这些差异除了实验时的差异外,抑制素A的检测方法的准确性现在还没有一个真正的金标准,并且在从组织中提取的和重组体的纯化都存在一定的不均一性,所以还需要有更特异,更敏感的检测方法并且使检测的指标标准化。另外,我国用于检测抑制素A的ELISA试剂盒也仍多是引进国外的进口产品,但是这样就大大增加了检测成本,阻碍了其在临床的普遍应用,而且在前期工作中我们已经研制出了AFP、HCG、UE3三种检测试剂盒,所以我们也希望能够研制出特异性强的国产化的检测试剂盒以便和上面三种指标联合应用,更好的推动唐氏综合症的临床检测。为此,本课题拟通过制备抗抑制素A的单克隆抗体,为进一步的唐氏综合症的筛查打下基础,进一步去评价抑制素A与在唐氏综合症诊断中的差异及价值,了解检测血清抑制素A是否在敏感性和特异性上优于其他的标志物。探讨其在唐氏综合症诊断中的可靠性和可行性。因为抑制素A在循环中不仅以二聚体的活性形式存在而且还会有许多未知功能的小分子形式,这就使得我们想找到一个能够针对它的更有特异性的检测方法。蛋白质的免疫原性主要是通过表位体现的,准确预测B细胞表位不仅有助于基础免疫学研究,也有助于疫苗和抗体的研究开发,有助于疾病的治疗与诊断。本研究一方面从胎盘中分别成功扩增并克隆出抑制素A的亚基α(INHA)的基因全长片段和其去信号肽的片段,将所得的目的基因分别克隆至pET-32a表达载体,发现只有其去信号肽的片段在大肠杆菌中有大量不可溶性表达。用亲和层析法纯化重组蛋白。INHA蛋白通过抗His的抗体鉴定后免疫小鼠制备多克隆抗体。分别用ELISA、Western-blot法检测和鉴定多克隆抗体。另一方面,本研究从抑制素A两个亚基的氨基酸序列入手用ExPASy,SignalP等软件分析该基因(GenBank号:NM 002191;NM 002192)编码蛋白综合分析预测INHA和INHBA的B细胞抗原位点,然后把氨基酸序列提交到NCBI中进行同源性比对,综合分别分析选取四条抗原表位并以固相合成法合成抗原。把纯化后的抗原连接大分子的蛋白后免疫小鼠制备多克隆和单克隆抗体。分别用ELISA、Western-blot法检测和鉴定抗体。结果显示,利用RT-PCR技术成功克隆了INHA的基因全长片段(INHA)和其去信号肽的片段(INHA1),经测序并与GenBank中序列进行比对,结果完全一致,大小分别为1101bp和1054bp。并且将INHA和INHA1基因分别克隆到pET-32a原核表达质粒,经PCR、限制性酶切分析和测序等鉴定,成功构建了pET32a-A和pET32a-A1重组质粒。重组质粒pET32a-A1在大肠杆菌中表达出INHA1融合蛋白,表达产物均主要以不可溶性形式存在。重组蛋白分子量约为58kDa与理论值基本符合。表达量分别约占菌体总蛋白的20%,重组蛋白经过纯化后,纯度均达80%。用抗His的抗体对表达产物进行Western-blot分析,发现特异性区带出现在58kDa处,表明该蛋白确实是所要表达的融合蛋白。进一步用纯化的表达产物免疫小鼠制备免疫血清,ELISA分析表明重组蛋白能与该免疫血清起反应,而与正常小鼠血清不发生交叉反应,Western-blot分析,发现可与胎盘中的天然蛋白反应,说明重组蛋白具有抗原活性。用纯化的表达产物INHA1以及免疫小鼠制备多克隆抗体,然后将抗多肽抗体与INHA1抗原以及多肽片段与抗INHA1的抗体进行反应,进一步筛选了一个优势的抗原表位并分别制备了24株单克隆抗体。其中五株单抗细胞株培养上清的效价用ELISA法检测,OD值可达到1:128到1:512之间。Western-blot结果显示:可与胎盘中的天然蛋白发生特异性反应,而对照组则无明显条带出现。说明制备的单抗均为特异性单抗。以上结果表明,我们已经成功地构建了pET32a-A1原核重组表达质粒,而且在大肠杆菌中大量表达出不溶性重组蛋白,经过复性获得具有活性的INHA1蛋白,免疫小鼠后获得多克隆抗体。另外,我们也成功的预测了抑制素A两个亚基的B细胞抗原表位,筛选并建立了多株单克隆抗体。这些都为抑制素A临床的应用打下了基础。
[Abstract]:Inhibin A is a (32 kDa) macromolecule glycoprotein peptide hormone consisting of two subunits of alpha and beta coupled with disulfide bond (-S-S) and belongs to the transforming growth factor beta (TGF2 beta) polypeptide family. Its basic function is to inhibit pituitary production of follicle stimulating hormone (FSH). Inhibin also regulates follicular fineness in gonads by autocrine and paracrine forms. Cell differentiation and steroid production; most circulating inhibin is produced by the gonad; moreover, they are expressed in many tissues outside the gonad, including brain enlargement, adrenal gland, bone marrow, placenta, and have a certain regulatory role locally. Inhibin A is not only related to follicular development and ovarian tumors, but also found in recent years as a second trimester of pregnancy. An indicator of Down's syndrome screening.
Down's syndrome (Down's) is the earliest and most common autosomal disease found in humans, and it is also one of the important causes of congenital mental retardation in children. Amniocentesis and chorionic villus biopsy are limited to high-risk groups, only 20% of Down's disease can be detected. In addition, both amniocentesis and chorionic villus biopsy are invasive examinations and not suitable for group examination. In order to minimize prenatal invasive examinations and reduce the birth rate of Down's disease, prenatal serological screening has been widely used in clinical practice. In the second trimester of pregnancy, serum levels of these markers overlap in both normal and abnormal groups. Therefore, the discovery of new markers and the application of a comprehensive test protocol are effective ways to improve the detection rate. Inhibin A has been used as an effective marker for Down syndrome in pregnant women whose serum levels are higher than those in normal pregnancies.
The mechanism of maternal serum marker concentration in Down's syndrome is still unclear, but the decrease of fetal products or fetal placental products (AFP, uE3) and the increase of placental products (HCG, DIA) confirm that inhibin A in maternal plasma mainly comes from placenta rather than from fetus. During pregnancy, serum levels of inhibin A in the form of active dimer (DIA) secreted mainly by the placenta increased significantly. During the first three months of pregnancy, the serum levels of DIA increased and then decreased around the tenth week, and remained stable throughout the fifteenth to twenty-fifth weeks of pregnancy until the end of pregnancy. This stable secretion cycle allows DIA testing to be optimized for some other prenatal diagnostic criteria. Since serum inhibin A levels in pregnant women with Down syndrome were detected to be twice or higher than those in normal pregnant women, DIA has been compared with other prenatal diagnostic markers and found to be pregnant. In the second trimester of pregnancy, AFP + HCG + UE3 can increase the detection rate by eight percentage points to 84%, and the false positive rate is only 5%. In addition, HCG content changes greatly during the 15-20 weeks of pregnancy and the relative level of DIA is relatively stable, so many scholars believe that the detection of DIA may be better than HCG. We all know that the gestational age is based on the last menstrual period. However, there may be errors in the results. Compared with other serum markers, DIA levels do not fluctuate significantly with gestational age in the second and third months of pregnancy, which has a significant advantage in explaining these results when studying more reliable data to assess Down syndrome and is not misrepresented. The exact age of pregnancy is affected.
With the current understanding of the dangers of Down's syndrome, it is hoped that early diagnosis and early termination of pregnancy can be achieved without risk. Therefore, laboratory screening with DIA combined with NT, PAPP-A, and beta-HCG can increase the early diagnosis rate to 90%.
Early detection of inhibin A is difficult to detect the free form of DI A and alpha subunit, so there are many uncertain values in the test results. However, with the emergence of specific antibodies against DI A, the detection of DI A is not difficult. Now the only method to determine the level of inhibin A Pick is still immunoassay. This method has been proven to be specific for the detection of DIA in human plasma. With the development of these techniques, the results of screening in different laboratories have also been greatly different. It has been reported that this method can be used in pregnant women with Down's disease and normal pregnant women. In addition to the experimental differences, the accuracy of the assay for inhibin A is not yet a true gold standard, and there is a certain degree of heterogeneity in the extraction and purification of recombinants from tissues, so more specificity is needed. In addition, most of the ELISA kits used to detect inhibin A in our country are imported products, but this greatly increases the cost of detection, hindering its widespread clinical application, and in the previous work we have developed three kinds of AFP, HCG, UE3. Detection kit, so we also hope to be able to develop a strong specificity of the domestic detection kit for the above three indicators in combination with the application, better promote the clinical detection of Down syndrome.
Therefore, we intend to prepare monoclonal antibodies against inhibin A to lay a foundation for further screening of Down syndrome, to further evaluate the difference and value of inhibin A in the diagnosis of Down syndrome, and to understand whether serum inhibin A is superior to other markers in sensitivity and specificity. Reliability and feasibility of diagnosis.
Because inhibin A exists not only as a dimer but also as a small molecule with many unknown functions in the cycle, we want to find a more specific detection method for inhibin A.
The immunogenicity of proteins is mainly expressed by epitopes. Accurate prediction of B cell epitopes is not only conducive to basic immunological research, but also conducive to the research and development of vaccines and antibodies, and is conducive to the treatment and diagnosis of diseases.
On the one hand, the full-length fragment of inhibin A subunit alpha (INHA) gene and the fragment of its desensitizing peptide were successfully amplified and cloned from placenta. The obtained target gene was cloned into pET-32a expression vector, and only the fragment of its desensitizing peptide was found to be highly insoluble in E.coli. The recombinant protein INHA was identified by anti-His antibody and then immunized mice to prepare polyclonal antibodies. The polyclonal antibodies were detected and identified by ELISA and Western-blot respectively. On the other hand, the amino acid sequences of the two subunits of inhibin A were analyzed by ExPASy and SignalP software (GenBank: NM 002191; NM 002192). The antigenic sites of INHA and INHBA were predicted by comprehensive analysis of coding proteins. The amino acid sequences were submitted to NCBI for homology comparison. Four antigenic epitopes were selected and synthesized by solid-phase synthesis. The purified antigen was linked to the macromolecule protein and the mice were immunized to prepare polyclonal and monoclonal antibodies. Do not use ELISA and Western-blot to detect and identify antibodies.
The results showed that the full-length fragment (INHA) of INHA gene and the fragment of its desensitizing peptide (INHA1) were successfully cloned by RT-PCR. The results were identical with those in GenBank. The INHA and INHA1 genes were cloned into pET-32a prokaryotic expression plasmids, and then PCR and restriction enzyme digestion were performed. PET32a-A and pET32a-A1 recombinant plasmids were successfully constructed by sequencing and sequencing.
The recombinant plasmid pET32a-A1 expressed INHA1 fusion protein in E.coli, and the expressed products were mainly insoluble. The molecular weight of the recombinant protein was about 58 kDa, which accounted for about 20% of the total bacterial protein, and the purity of the recombinant protein was up to 80% after purification. Estn-blot analysis showed that the specific band appeared at 58 kDa, indicating that the protein was indeed the fusion protein to be expressed. Further immunized mice with purified expression products to prepare immune serum. ELISA analysis showed that the recombinant protein could react with the immune serum, but did not cross-react with the normal mice serum. Western-blot analysis showed that the recombinant protein could not react with the normal mice serum. It is found that it can react with natural protein in placenta, indicating that the recombinant protein has antigenic activity.
Polyclonal antibodies were prepared with purified expression product INHA1 and immunized mice. Anti-polypeptide antibodies were reacted with INHA1 antigen and polypeptide fragments with anti-INHA1 antibodies. A dominant antigen epitope was further screened and 24 monoclonal antibodies were prepared respectively. The titer of supernatant of five monoclonal antibodies was determined by ELISA. The OD value was 1:128 to 1:512. Western-blot analysis showed that it could react specifically with the natural protein in placenta, but no obvious band appeared in the control group.
These results indicate that we have successfully constructed the recombinant plasmid pET32a-A1, and expressed a large number of insoluble recombinant proteins in E. Epitopes screened and established multiple monoclonal antibodies, which laid the foundation for clinical application of inhibin A.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

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