【摘要】：抑制素A是一種(32kDa)大分子糖蛋白肽類(lèi)激素,由α及β兩個(gè)亞基以二硫鍵(—S—S)偶聯(lián)構(gòu)成,屬于轉(zhuǎn)化生長(zhǎng)因子β(TGF2β)多肽家族。其基本作用是抑制垂體產(chǎn)生卵泡刺激素(FSH),卵泡抑制素也在性腺中通過(guò)自分泌和旁分泌的形式調(diào)節(jié)卵泡細(xì)胞的分化和甾體的生成;大多數(shù)循環(huán)抑制素由性腺產(chǎn)生,另外它們?cè)谠S多性腺外的組織,包擴(kuò)腦,腎上腺,骨髓,胎盤(pán)都有表達(dá)并且在局部都有一定的調(diào)節(jié)作用。抑制素A不僅與卵泡發(fā)育和卵巢腫瘤有關(guān)而且近年來(lái)還發(fā)現(xiàn)它可以作為孕中期唐氏綜合癥篩查的一項(xiàng)指標(biāo)。 唐氏綜合癥(Down's)是人類(lèi)最早發(fā)現(xiàn)且最常見(jiàn)的常染色體病,也是造成兒童先天性智力低下的重要原因之一。唐氏無(wú)明顯的家族史,每個(gè)孕婦都有生出該患兒的可能性,至今尚無(wú)有效的治療手段,所以產(chǎn)前診斷就顯得較為重要,目前開(kāi)展的羊水穿刺及絨毛活檢僅限于高危人群,僅能檢出20%的唐氏患兒,此外羊水穿刺及絨毛活檢均為侵入性檢查,不適于群體檢查,為了能最大限度減少產(chǎn)前有創(chuàng)檢查降低唐氏患兒的的出生率,產(chǎn)前的血清學(xué)的篩查已被廣泛用于臨床。現(xiàn)在妊娠中期的血清學(xué)檢測(cè)主要采取AFP+HCG+UE3的三聯(lián)實(shí)驗(yàn),但是這些標(biāo)志物的血清水平在正常和非正常人群都有所重疊,所以,新的標(biāo)志物的發(fā)現(xiàn)和應(yīng)用綜合的檢測(cè)方案是提高檢出率的有效的方法。自從發(fā)現(xiàn)抑制素A的血清水平在懷有唐氏患兒的孕婦的血清中的水平高于正常妊娠后,抑制素A已被作為檢測(cè)唐氏綜合癥的一個(gè)有效的標(biāo)志物。 Down's綜合征妊娠中母血標(biāo)記物濃度變化機(jī)制尚不清楚,但胎兒產(chǎn)物或胎兒胎盤(pán)產(chǎn)物(AFP,uE3)減少,而胎盤(pán)產(chǎn)物(HCG,DIA)增加,證實(shí)了母體血漿中抑制素A等成分主要來(lái)自胎盤(pán)而不是胎兒。抑制素A作為Down's綜合征孕中期血清篩查的第4個(gè)標(biāo)記物,在妊娠時(shí),抑制素A主要由胎盤(pán)分泌并且有活性的二聚體的形式(DIA)在血液中的水平明顯升高。妊娠前三個(gè)月中DIA的血清水平升高隨后大約在第10周下降,在第15-25周期間產(chǎn)婦的血清中的水平保持一個(gè)穩(wěn)定的水平一直持續(xù)到妊娠末期達(dá)到峰值。它的這種穩(wěn)定分泌周期使DIA的檢測(cè)更加優(yōu)化于其它的一些產(chǎn)前診斷指標(biāo)。自從檢測(cè)到在懷有唐氏綜合癥的產(chǎn)婦血清中抑制素A的水平高于正常孕婦的兩倍或更高,DIA已經(jīng)和其他產(chǎn)前診斷的標(biāo)志物都作了比較和對(duì)照,發(fā)現(xiàn)在妊娠的中期聯(lián)合AFP+HCG+UE3,可以將檢出率提高八個(gè)百分點(diǎn)達(dá)到84%,并且假陽(yáng)性率只有5%。另外HCG在妊娠15-20周含量變化較大而相對(duì)的DIA的水平卻相對(duì)穩(wěn)定,所以有許多學(xué)者認(rèn)為DIA的檢測(cè)可能更優(yōu)于HCG。我們都知道妊齡多是根據(jù)末次月經(jīng)所得。但是這樣所得的結(jié)果可能存在誤差,和其他血清標(biāo)志物相比較,DIA的水平在妊娠的第二和第三個(gè)月不會(huì)隨著妊齡的變化而有很大的波動(dòng),這在研究更可靠的數(shù)據(jù)去評(píng)價(jià)唐氏綜合癥時(shí),它有很大的優(yōu)勢(shì)去解釋這些結(jié)果,并且不會(huì)被不正確的妊齡所影響。 隨著現(xiàn)在人們對(duì)唐氏綜合癥的危害的了解,大家都希望能在沒(méi)有危險(xiǎn)的情況下進(jìn)行早期診斷,早期終止妊娠。所以,又有實(shí)驗(yàn)室用DIA聯(lián)合NT,PAPP-A,β—HCG進(jìn)行了篩查,當(dāng)結(jié)合年齡若加上DIA則可以使早期診斷率提高到90%。 早期抑制素A的測(cè)定在檢測(cè)DIA和α亞單位的游離形式之間存在一定困難,因此在檢測(cè)結(jié)果中出現(xiàn)了許多不確定的值,但是隨著針對(duì)DIA的特異性抗體的出現(xiàn)使得DIA的檢測(cè)不在是難事�，F(xiàn)在唯一能測(cè)定抑制素A皮克水平的方法仍是免疫測(cè)定法。這種方法都已被證實(shí)可以用來(lái)特異性的檢測(cè)人類(lèi)血漿中DIA的水平,這種方法都是雙夾心酶聯(lián)免疫吸附實(shí)驗(yàn)隨著這些檢測(cè)技術(shù)的發(fā)展,使得各個(gè)實(shí)驗(yàn)室的篩查結(jié)果也有了很大的差異,有報(bào)道說(shuō)在比較懷有唐氏的孕婦和正常的孕婦抑制素A的水平時(shí)發(fā)現(xiàn)在7-8周時(shí)其水平降低而在9-11周時(shí)升高。這些差異除了實(shí)驗(yàn)時(shí)的差異外,抑制素A的檢測(cè)方法的準(zhǔn)確性現(xiàn)在還沒(méi)有一個(gè)真正的金標(biāo)準(zhǔn),并且在從組織中提取的和重組體的純化都存在一定的不均一性,所以還需要有更特異,更敏感的檢測(cè)方法并且使檢測(cè)的指標(biāo)標(biāo)準(zhǔn)化。另外,我國(guó)用于檢測(cè)抑制素A的ELISA試劑盒也仍多是引進(jìn)國(guó)外的進(jìn)口產(chǎn)品,但是這樣就大大增加了檢測(cè)成本,阻礙了其在臨床的普遍應(yīng)用,而且在前期工作中我們已經(jīng)研制出了AFP、HCG、UE3三種檢測(cè)試劑盒,所以我們也希望能夠研制出特異性強(qiáng)的國(guó)產(chǎn)化的檢測(cè)試劑盒以便和上面三種指標(biāo)聯(lián)合應(yīng)用,更好的推動(dòng)唐氏綜合癥的臨床檢測(cè)。 為此,本課題擬通過(guò)制備抗抑制素A的單克隆抗體,為進(jìn)一步的唐氏綜合癥的篩查打下基礎(chǔ),進(jìn)一步去評(píng)價(jià)抑制素A與在唐氏綜合癥診斷中的差異及價(jià)值,了解檢測(cè)血清抑制素A是否在敏感性和特異性上優(yōu)于其他的標(biāo)志物。探討其在唐氏綜合癥診斷中的可靠性和可行性。 因?yàn)橐种扑谹在循環(huán)中不僅以二聚體的活性形式存在而且還會(huì)有許多未知功能的小分子形式,這就使得我們想找到一個(gè)能夠針對(duì)它的更有特異性的檢測(cè)方法。 蛋白質(zhì)的免疫原性主要是通過(guò)表位體現(xiàn)的,準(zhǔn)確預(yù)測(cè)B細(xì)胞表位不僅有助于基礎(chǔ)免疫學(xué)研究,也有助于疫苗和抗體的研究開(kāi)發(fā),有助于疾病的治療與診斷。 本研究一方面從胎盤(pán)中分別成功擴(kuò)增并克隆出抑制素A的亞基α(INHA)的基因全長(zhǎng)片段和其去信號(hào)肽的片段,將所得的目的基因分別克隆至pET-32a表達(dá)載體,發(fā)現(xiàn)只有其去信號(hào)肽的片段在大腸桿菌中有大量不可溶性表達(dá)。用親和層析法純化重組蛋白。INHA蛋白通過(guò)抗His的抗體鑒定后免疫小鼠制備多克隆抗體。分別用ELISA、Western-blot法檢測(cè)和鑒定多克隆抗體。另一方面,本研究從抑制素A兩個(gè)亞基的氨基酸序列入手用ExPASy,SignalP等軟件分析該基因(GenBank號(hào):NM 002191;NM 002192)編碼蛋白綜合分析預(yù)測(cè)INHA和INHBA的B細(xì)胞抗原位點(diǎn),然后把氨基酸序列提交到NCBI中進(jìn)行同源性比對(duì),綜合分別分析選取四條抗原表位并以固相合成法合成抗原。把純化后的抗原連接大分子的蛋白后免疫小鼠制備多克隆和單克隆抗體。分別用ELISA、Western-blot法檢測(cè)和鑒定抗體。 結(jié)果顯示,利用RT-PCR技術(shù)成功克隆了INHA的基因全長(zhǎng)片段(INHA)和其去信號(hào)肽的片段(INHA1),經(jīng)測(cè)序并與GenBank中序列進(jìn)行比對(duì),結(jié)果完全一致,大小分別為1101bp和1054bp。并且將INHA和INHA1基因分別克隆到pET-32a原核表達(dá)質(zhì)粒,經(jīng)PCR、限制性酶切分析和測(cè)序等鑒定,成功構(gòu)建了pET32a-A和pET32a-A1重組質(zhì)粒。 重組質(zhì)粒pET32a-A1在大腸桿菌中表達(dá)出INHA1融合蛋白,表達(dá)產(chǎn)物均主要以不可溶性形式存在。重組蛋白分子量約為58kDa與理論值基本符合。表達(dá)量分別約占菌體總蛋白的20%,重組蛋白經(jīng)過(guò)純化后,純度均達(dá)80%。用抗His的抗體對(duì)表達(dá)產(chǎn)物進(jìn)行Western-blot分析,發(fā)現(xiàn)特異性區(qū)帶出現(xiàn)在58kDa處,表明該蛋白確實(shí)是所要表達(dá)的融合蛋白。進(jìn)一步用純化的表達(dá)產(chǎn)物免疫小鼠制備免疫血清,ELISA分析表明重組蛋白能與該免疫血清起反應(yīng),而與正常小鼠血清不發(fā)生交叉反應(yīng),Western-blot分析,發(fā)現(xiàn)可與胎盤(pán)中的天然蛋白反應(yīng),說(shuō)明重組蛋白具有抗原活性。 用純化的表達(dá)產(chǎn)物INHA1以及免疫小鼠制備多克隆抗體,然后將抗多肽抗體與INHA1抗原以及多肽片段與抗INHA1的抗體進(jìn)行反應(yīng),進(jìn)一步篩選了一個(gè)優(yōu)勢(shì)的抗原表位并分別制備了24株單克隆抗體。其中五株單抗細(xì)胞株培養(yǎng)上清的效價(jià)用ELISA法檢測(cè),OD值可達(dá)到1:128到1:512之間。Western-blot結(jié)果顯示:可與胎盤(pán)中的天然蛋白發(fā)生特異性反應(yīng),而對(duì)照組則無(wú)明顯條帶出現(xiàn)。說(shuō)明制備的單抗均為特異性單抗。 以上結(jié)果表明,我們已經(jīng)成功地構(gòu)建了pET32a-A1原核重組表達(dá)質(zhì)粒,而且在大腸桿菌中大量表達(dá)出不溶性重組蛋白,經(jīng)過(guò)復(fù)性獲得具有活性的INHA1蛋白,免疫小鼠后獲得多克隆抗體。另外,我們也成功的預(yù)測(cè)了抑制素A兩個(gè)亞基的B細(xì)胞抗原表位,篩選并建立了多株單克隆抗體。這些都為抑制素A臨床的應(yīng)用打下了基礎(chǔ)。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392.1

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