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目的:利用体内噬菌体展示技术筛选并鉴定紫癜性肾炎肾组织特异性结合肽。方法:构建大鼠紫癜性肾炎模型。尾静脉注射噬菌体展示环七肽库,筛选与紫癜性肾炎肾组织特异性结合的噬菌体,经过3轮体内筛选后,提取阳性单克隆噬菌体,并进行DNA测序。利用ELISA方法鉴定单克隆噬菌体对紫癜性肾炎的结合。通过化学合成的方法合成筛选获得的多肽,以流式细胞检测其与肾细胞的结合能力。同时,以合成的多肽体内封闭,检测噬菌体与合成多肽竞争结合紫癜性肾炎肾组织的能力。结果:成功筛选出于大鼠紫癜性肾炎肾组织结合的阳性噬菌体。在随机挑选的43个单克隆噬菌斑中,挑选了其中16个亲和力高的噬菌体,测序后获得了紫癜性肾炎的结合肽CQGPWKLTC。流式结果表明筛选的多肽能够与肾细胞特异性结合。获得的阳性噬菌体能够与紫癜性肾炎肾组织特异性结合,并且与体外合成的多肽具有竞争结合能力。结论:利用体内噬菌体展示技术筛选了与紫癜性肾炎肾组织特异性结合的肽CQGPWKLTC。
OBJECTIVE: To screen and identify renal tissue-specific binding peptides of purpuric nephritis by phage display in vivo. Methods: To establish a rat model of purpuric nephritis. The phage displayed the heptapeptide library by tail vein injection, and screened the phage that specifically binds to the kidney tissue of purpuric nephritis. After 3 rounds of in vivo screening, positive monoclonal phage were extracted and sequenced. The binding of monoclonal phage to purpuric nephritis was identified by ELISA. The synthesized peptides were synthesized and screened by chemical synthesis, and their binding ability to renal cells was detected by flow cytometry. At the same time, the synthesized peptides were blocked in vivo, and the ability of phage and synthetic peptides to compete for binding to renal tissue of purpuric nephritis was tested. Results: The positive phage derived from kidney tissue of purpuric nephritis in rats were successfully screened. Of the 43 monoclonal plaques randomly selected, 16 were high-affinity phage, and sequenced to obtain the binding peptide CQGPWKLTC of purpuric nephritis. The flow cytometry results showed that the selected peptides could specifically bind with renal cells. The obtained positive phage can specifically bind to purpuric nephritis kidney tissue and have competitive binding ability with the synthesized polypeptide in vitro. Conclusion: The in vivo phage display technique was used to screen CQGPWKLTC, a peptide that specifically binds to renal tissue of purpuric nephritis.