【摘要】：肝細胞肝癌(HCC),簡稱肝癌,是惡性程度極高的惡性腫瘤,素有“癌王”之稱,目前臨床對肝癌的診治仍然十分困難,仍以手術治療和放化療為主。近年來,以抗體為基礎的導向治療方法的研究為腫瘤的診治帶來了生機,先后出現了以單鏈抗體、二硫鍵抗體、Fab抗體等抗體形式為導向載體,以細胞因子、毒素分子、超抗原、放射性核素為“彈頭”的導向治療制劑。 在單鏈抗體、二硫鍵抗體、Fab抗體這幾種導向載體中,二硫鍵抗體、Fab抗體因重鏈和輕鏈之間有二硫鍵相連,穩(wěn)定性較高,但表達困難或較難獲得較高產量;單鏈抗體有較成熟的原核表達系統(tǒng),產量較高,但穩(wěn)定性較差。為了解決這一問題,我們構建了抗肝癌單鏈二硫鍵穩(wěn)定抗體(這一新型抗體形式)靶向超抗原SEA(D227A)分子,以提高抗肝癌抗體靶向超抗原SEA(D227A)分子的產量和穩(wěn)定性。單鏈二硫鍵穩(wěn)定抗體(single-chain disulfide-stabilized Fv,scdsFv)是在單鏈抗體的基礎上,在抗體的重鏈可變區(qū)和輕鏈可變區(qū)的框架區(qū)分別突變出一個半胱氨酸,以完整的一條鏈形式表達,這樣可在輕、重鏈可變區(qū)之間形成二硫鍵,以達到提高穩(wěn)定性和產量的目的。 本研究通過連接肽將抗肝癌二硫鍵穩(wěn)定抗體的VH、VL連接起來,構建了單鏈二硫鍵穩(wěn)定抗體,這一最具應用前景的Fv抗體形式,并將其同超抗原SEA(D227A)融合,構建了抗肝癌scdsFv-SEA(D227A)重組表達質粒,以期提高產量和穩(wěn)定性。 ScdsFv-SEA(D227A)重組表達質粒經IPTG誘導后,在大腸桿菌BL21plusS中以包涵體形式表達,表達量占菌體蛋白的30%左右。包涵體經充分洗滌和變性、復性后,用Q Sepharose HP和Hiprep 26/60 Sephacryl S-200 HR進行純化,純度達90%以上,BCA蛋白定量顯示每升誘導菌液的產量高達60mg,提高了產量。
[Abstract]:Hepatocellular carcinoma (HCC),) is a malignant tumor with a high degree of malignancy. It is known as the "king of cancer". At present, the diagnosis and treatment of HCC is still very difficult, and it is still dominated by surgical treatment and radiotherapy and chemotherapy. In recent years, the study of antibody-based directed therapy has brought vitality to the diagnosis and treatment of tumors. Single chain antibody, disulfide bond antibody, Fab antibody and other antibodies have been used as guiding vectors, with cytokines and toxin molecules. Superantigens, radionuclides are guided therapeutic agents for warheads. In the vector of single chain antibody, disulfide bond antibody and Fab antibody are more stable because of the connection of disulfide bond between heavy chain and light chain, but the expression is difficult or difficult to obtain higher yield. Single chain antibody has a mature prokaryotic expression system, the yield is high, but the stability is poor. In order to solve this problem, we constructed a single chain disulfide stable antibody (D227A) targeting superantigen SEA (D227A) molecule to improve the yield and stability of anti-HCC antibody targeting superantigen SEA (D227A) molecule. Single chain disulfide stable antibody (single-chain disulfide-stabilized Fv,scdsFv) is based on single chain antibody, which mutates a cysteine in the frame region of the heavy chain variable region and the light chain variable region of the antibody, respectively, and expresses it in the form of a complete chain. In this way, disulfide bonds can be formed between the variable regions of light and heavy chains in order to improve the stability and yield. In this study, the single chain disulfide stable antibody, the most promising form of Fv antibody, was constructed by ligating peptide to ligate the VH,VL of anti-HCC disulfide stable antibody, and it was fused with superantigen SEA (D227A). The recombinant expression plasmid of anti-HCC scdsFv-SEA (D227A) was constructed in order to improve the yield and stability. After the recombinant expression plasmid ScdsFv-SEA (D227A) was induced by IPTG, it was expressed in the form of inclusion body in E. coli BL21plusS, which accounted for about 30% of the bacterial protein. After full washing and denaturation, the inclusion body was purified with Q Sepharose HP and Hiprep 26 鈮，

本文編號：2487613