摘要
壬基酚(Nonylphenol,NP)是已经确认的环境内分泌干扰物之一,且在环境中的污染状况不容乐观,对其进行监测和评估是环境监测和治理的一项重要内容。使用仪器进行检测虽然准确,但费时、费力、成本高。而快速生物检测技术如免疫学检测的基础是需要有该小分子污染物的特异抗体。基因工程重组单克隆抗体(recombination monoclonal antibody,RMA)是90年代以来,人们利用基因工程重组技术,有目的地在基因水平上对抗体分子进行切割、拼接或修饰,或者直接合成基因序列,再将重组DNA或重组蛋白基因导入细胞表达产生的一类抗体,其化学结构特点与单克隆抗体相同,具有稳定的免疫特异性。其中单链抗体(singe chain variable fragment,ScFv)由于低或无免疫原性、分子量小、组织穿透力强,成本低,可大规模生产等特点已应用于医学领域。单链抗体可以通过噬菌体展示技术或核糖体展示技术加以制备。其中,核糖体展示技术完全在体外进行,库容量远远大于噬菌体展示文库,此外核糖体展示技术具有简便的建库和筛选方法,勿需选择压力,通过引入突变和重组技术来提高靶标蛋白的亲和力。上述优点都使核糖体展示技术显示出了诱人的发展前景。
本实验利用核糖体展示抗体库库容量大(可达1013-1015)的优势与天然抗体库的通用性,以环境内分泌干扰物壬基酚的抗原(NP-BSA)为靶标,从未经免疫的不同品系(Balb/C、C57)小鼠的脾细胞中提取总RNA,反转录合成cDNA第一链后,设计多对适当引物扩增VH、VL基因片段,构建的单链抗体文库重链H和轻链L大小都为350bp左右,设计引物从载体PT7PD扩增出间隔序列P(大小为294bp),核糖体展示元件T(大小为93bp)采用全基因合成方法得到。以重叠引物延伸法将这几段序列拼接,先将核糖体展示元件T与重链H连接,间隔序列P与轻链L连接,分别纯化回收后再经linker(Gly3Ser)4连接,将TH和LP连接成全长ScFv片段,大小约为1100bp,从而构建了核糖体展示鼠源天然抗体基因库,体外鉴定该天然抗体基因库的转录活性、反转录活性和蛋白质水平的活性。
将构建的单链抗体基因文库进行体外转录和体外翻译后,产生了单链抗体-核糖体-mRNA(ARM)三联复合体文库。以小分子抗原壬基酚为靶标,利用固相亲和方法筛选该三联复合体,洗涤后,通过改变Mg2+浓度将筛选得到的ARM复合物解离,得到相应的mRNA,经过RT-PCR得到筛选后的DNA文库。通过多轮生物淘筛,抗原阳性的单链抗体得到富集,最终获得高特异性抗壬基酚的抗体。将该抗体序列在大肠杆菌中进行高效表达并进行初步鉴定,因为所用天然抗体库本身具有的通用性,理论上可以以任意抗原为靶标筛选其亲和抗体,这就为利用核糖体展示技术从天然抗体库中筛选多种抗原特别是小分子半抗原的特异性抗体奠定了基础。
As one of the Environmental Endocrine Disrupting Chemicals(EDCs), monitoring and evaluation of Nonylphenol is importent in area survey.Although it is accurate by using instrument,these methods are time-consuming, laborious and costly. The rapid biological detection technologies such as immunological detection is based on the specific antibody of the small molecule pollutants. Recombination monoclonal antibody(RMA), since the 1990s, is a kind of antibody that antibody gene is reformed by cutting, splicing,modified, or synthesisised directly, then transformed into living cells for expression. Its chemical constitution is same as the monoclonal antibody, and remins stable immune specificity. Among all of these engineering antibodies, single chain variable fragment (ScFv) has been used in medical domain with characters of low or no immunogenicity, small molecular weight, powerful tissue penetration, low cost, full scale operation and so on. ScFvs can be obtained by phage display, ribosome display etc.. Because of entirely in vitro, ribosome display is far greater than phage display library in library capicity, and is simple in construction and screening with no selection pressure, which would enhance the affinity of targeting protein by the introduction of mutation and recombination. These advantages have made ribosome display technology demonstrate the attractive prospects.
Utilizing the large capacity (up to 1013-1015) and the versatility of native antibody library of ribosome display, we extracted the total RNA from the mouse in different stains(Balb/C, C57) and synthetized the first chain cDNA by reverse transcription. After designing more appropriate primers to amplify the VH and VL gene fragments(about 350 bp) ,ribosomal display elements T (93 bp) was synthesized in vitro and interval sequence P (294 bp) from the vector PT7PD, we spliced these four fragments by overlap extention PCR. Firstly, ribosome display components T and heavy chain H were spliced;secondly, interval sequence P and light chain L were spliced, at last, the fragment TH and LP were spliced by the linker (Gly3Ser) 4 after the purification respectively and Ribosome display antibody library have been constructed which was about 1100bps then we identificated the activity of transcription, reverse transcription and translation of this library in vitro.
After transcription and translation of the ScFvs library in vitro, we got the antibody-ribosome-mRNA(ARM)complex. Targeted by nonylphenol(NP-BSA), this complex was screened by solid affinity selection. We degraded the concentration of Mg2+ to elute mRNA which was used for in reverse transcription to create DNA library for next round. After several rounds of selection. specific antibody was finally obtained.The antibody was expressed in Escherichia coli and then purified. Because the versatility of native antibody library, theoretically specific antibody can be selected for any kinds of antigen using as targets, This methods established the foundation that we can select specific antibodies of any antigens especially micromolecule haptens.
引文
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