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本研究从哮喘患者外周血分离淋巴细胞、提取总mRNA,采用RT-PCR技术分别构建重链可变区VH(variable region of heavy chain)和轻链可变区VL(variable region of light chain)cDNA库,然后通过连接肽(Gly4Ser)3和特定引物将VH和VL cDNA基因组装成人源单链抗体(scFv)核糖体展示模板基因库。应用兔网织红细胞核糖体展示技术,单引物原位反转录技术,经3轮展示富集并回收针对人源IgE蛋白(免疫球蛋白E)的目的单链抗体基因;回收的抗体基因经双酶切后与pET22b(+)载体连接,转化至E.coli Rosseta(DE3)宿主细胞,应用菌落PCR结合Dot blotting技术快速鉴定阳性克隆,抗原ELISA法进一步鉴定阳性克隆。VH和VL基因库得到正确的构建,长度分别为400和710 bp,库容为1013。核糖体展示模板构建正确,长度为1 100 bp。该基因库经过3轮针对IgE蛋白的核糖体展示,目的基因得到了有效富集和回收。经过高效克隆、表达和鉴定,确定1株针对人IgE蛋白显示最强亲和力的阳性克隆菌pET-IgE-6。经测序和序列分析证实该单链抗体为人源抗体,序列未见国内外报道。结果表明,采用患者外周血构建人源抗体基因库,结合核糖体展示技术,可以成功获得高亲和力的人源单链抗体分子。该技术路线为快速获得具有药用价值的人源抗体提供了参考。
In this study, lymphocytes were isolated from the peripheral blood of asthmatic patients and the total mRNA was extracted. The variable region of light chain (VH) and the variable region of light chain (VL) were constructed by RT-PCR. Library, and then the VH and VL cDNA genes were assembled into a human scFv ribosomal display template gene bank by ligating the peptide (Gly4Ser) 3 with the specific primers. The rabbit reticulocyte ribosome display technology and single-primer in situ reverse transcription technology were used to enrich and recover the single-chain antibody gene against human IgE protein (immunoglobulin E) after 3 rounds of display. The recovered antibody gene After digested with pET22b (+) vector, the recombinant plasmid was transformed into E.coli Rosseta (DE3) host cells. The positive clones were identified by colony PCR and Dot blotting. The positive clones were further identified by ELISA. The VH and VL gene libraries were correctly constructed, with lengths of 400 and 710 bp, respectively, and a capacity of 1013. The ribosome display template was correctly constructed and was 1 100 bp in length. After 3 rounds of ribosome display for IgE protein, the gene pool was effectively enriched and recovered. After efficient cloning, expression and identification, a positive clone strain pET-IgE-6 that showed the highest affinity for human IgE protein was identified. Sequencing and sequence analysis confirmed that the single chain antibody was human antibody, the sequence was not reported at home and abroad. The results showed that using human peripheral blood to construct human antibody gene bank and combining with ribosome display technology, high affinity human scFv molecules can be successfully obtained. The technical route provides a reference for rapidly obtaining human antibody with medicinal value.