抗Aβ人源單鏈抗體基因的篩選和全抗體基因的構建及表達研究
發(fā)布時間：2018-09-11 21:46
【摘要】： 阿爾茨海默病(Alzheimer disease,AD)是一種以老年人中進行性記憶障礙和智能衰退為主要臨床特征的中樞神經系統(tǒng)退行性疾病。老年斑(senile plaque,SP)、神經元纖維纏結(neurofibrillary tangles,NFT)和神經突觸減少或神經元丟失是AD的主要病理特征。其中老年斑的主要成分是β-淀粉樣多肽(beta-amyloidpeptide,Aβ)聚集形成的纖維,而Aβ纖維被認為是導致神經元損傷及認知功能衰退的主要致病物質。針對Aβ多肽在發(fā)病中的關鍵作用,研究如何阻止Aβ多肽聚集及清除大腦病變區(qū)已形成的Aβ多肽纖維已成為研究AD治療的前沿領域,抗Aβ多肽抗體治療AD也成為AD研究中的一個熱點。動物試驗已證明抗Aβ多肽抗體有清除小鼠大腦中Aβ纖維斑塊防治AD的作用,但至今尚未見人源化抗體有臨床應用的報道。 本課題研究從人源噬菌體單鏈抗體庫中篩選針對Aβ的單鏈抗體可變區(qū)(scFv)基因,并以基因工程構建人源抗Aβ全抗體的真核表達體系。主要包括兩部分:第一,從構建的人源噬菌體單鏈抗體庫中篩選抗Aβ的scFv基因,在原核細胞中進行可溶性表達,并進行抗體結合活性和生物學活性檢測;第二,重組IgG全抗體輕重鏈基因及全抗體單鏈基因,構建真核表達體系,進行真核細胞表達的初步研究。 一、抗Aβ人源scFv基因的篩選、原核表達與鑒定 以Aβ1-42多肽為抗原對抗體庫進行4輪富集篩選:用輔助噬菌體M13K07感染E.coli TG1轉化菌,展示出噬菌體形式的抗體庫。將此抗體庫加入到用Aβ1-42多肽包被的96孔板內,孵育一段時間后洗滌,能夠與抗原特異結合的scFv噬菌體克隆將被保留在96孔板內壁,然后用pH 2.2的甘氨酸-HCl緩沖液將特異結合的噬菌體洗脫下來,經pH8.0的Tris-HCl中和后,感染處于對數(shù)生長期的E.coli TG1,以擴增抗原特異性噬菌體克隆。這樣,經過4輪“吸附-洗脫-擴增”的富集過程,抗體庫中與Aβ1-42多肽特異性結合的噬菌體克隆從第1輪的3.31×10~(-4)增加到第4輪的7.65×10~(-2),得到了高度富集,多克隆噬菌體Elisa檢測也顯示了明確的富集效果。隨機挑取80個克隆,用M13K07感染后使scFv展示于噬菌體顆粒表面,ELISA篩選展示有抗Aβ1-42多肽scFv的噬菌體克隆。檢測結果顯示,得到7個陽性克隆,其中4個克隆ELISA檢測A值高于陰性對照5倍以上,另外3個在2.5～3倍之間。所有陽性克隆均與無關抗原BSA無交叉反應。對其中A值最高的2個克隆(A10、F2)的噬菌體顆粒上清感染E.coli HB2151,30℃,IPTG誘導表達20h,分別制備胞周質、培養(yǎng)基上清和全細胞提取物。經12%SDS-PAGE和western blot分析,目的蛋白相對分子量為33kD,主要集中于全細胞提取物,表達量占全菌蛋白60%左右。表達抗體ELISA檢測上清中和全菌蛋白中A值分別高于陰性對照5倍以上,在胞周質中高于2倍以上。抗體與Aβ1-40也有結合反應,但與BSA無交叉反應,顯示scFv抗體對Aβ具有特異性,結合位點主要在Aβ的N端。表達抗體的生物學活性鑒定示,可與人腦病變組織內淀粉樣斑塊中的抗原物質Aβ纖維結合,證明表達的scFv抗體可以應用于體內生物學效應實驗。將A10、F2克隆菌測序,用blast分析得到的scFv基因序列,從中分別確定了VH與VL的DNA序列,推導得到的氨基酸序列具有典型的抗體可變區(qū)結構。2個克隆的基因序列一致,表明2個克隆菌可能來源于同一株原始噬菌粒。 二、抗Aβ人源全抗體基因的重組構建、真核表達研究 首先對前期獲得的A10克隆scFv基因進行NCBI blast比對,獲得VH和VL基因相應的成熟信號肽基因,并合成信號肽基因,再設計特異引物分別擴增A10克隆的VH、VL、scFv基因和人源IgG的CH、CL、FC段基因,并利用重組PCR(overlap PCR)方法重組IgG全抗體重鏈基因(H信號肽基因+VH基因+CH基因)、IgG全抗體輕鏈基因(L信號肽基因+VL基因+CL基因)和全抗體單鏈基因(H信號肽基因+scFv基因+FC段基因),然后分別克隆到pcDNA3.1真核表達載體內。菌液PCR、雙酶切鑒定和DNA測序結果均顯示,重組的三種基因片段大小與預期一致,基因序列與預期的序列完全一致,讀碼框架和插入真核表達載體的方向完全正確。將IgG全抗體重鏈基因-載體質粒和IgG全抗體輕鏈基因-載體質粒共轉染CHO細胞,表達IgG全抗體;將全抗體單鏈基因-載體質粒轉染CHO細胞,表達單鏈全抗體(scFv抗體+FC段)。兩種轉染細胞初步的表達產物ELISA結果未見表達上清與Aβ1-42有陽性反應,后續(xù)工作仍需優(yōu)化細胞表達條件。現(xiàn)有的工作進展為今后進一步優(yōu)化表達、動物試驗及應用研究奠定基礎。 目前,雖然國內外已有幾家篩選到人源抗Aβ抗體的報道,但都仍處于實驗室研究階段,也未見全抗體或進入臨床試驗的報道。我們所得到的抗Aβ序列經過比對,與現(xiàn)有已發(fā)表的抗Aβ抗體序列均不同,表明是一個新的抗Aβ抗體�，F(xiàn)已在國內申報了專利,同時已完成了全抗體真核表達體系構建工作,后續(xù)的優(yōu)化表達工作正在進行。本課題研究工作的結果有利于今后進一步開展對AD的免疫治療和臨床應用研究。
[Abstract]:Alzheimer disease (AD) is a degenerative disease of the central nervous system characterized by progressive memory impairment and mental decline in the elderly. Senile plaque (SP), neurofibrillary tangles (NFT) and neurosynapse loss are the main pathological features of AD. Sign. The main component of senile plaque is the fibers formed by the aggregation of beta-amyloid peptide (A beta), which is thought to be the main pathogenic substance leading to neuronal damage and cognitive decline. Anti-A-beta polypeptide antibody has been proved to be effective in clearing the plaque of A-beta fibers in the brain of mice to prevent and treat AD.
In this study, we screened the variable region (scFv) gene of single-chain antibody against A beta from human phage single-chain antibody library and constructed the eukaryotic expression system of human anti-A beta whole antibody by genetic engineering. Soluble expression and detection of antibody binding activity and biological activity; Secondly, recombinant IgG antibody light and heavy chain gene and antibody single chain gene, construct eukaryotic expression system, and preliminary study of eukaryotic expression.
I. screening, prokaryotic expression and identification of human A scFv gene
Four rounds of enrichment and screening of the antibody library against A-beta-1-42 polypeptide were carried out. E.coli TG1 transformed strain was infected with auxiliary phage M13K07 and phage-like antibody library was displayed. The phage library was added to 96-well plate coated with A-beta-42 polypeptide and washed after incubation for a period of time. The scFv phage clone which could specifically bind to the antigen was retained. After neutralization with Tris-HCl at pH 8.0, E.coli TG1, which was in logarithmic growth phase, was infected to amplify antigen-specific phage clones. Thus, after four rounds of "adsorption-elution-amplification" enrichment, the antibody library was specific to the peptide A-beta 1-42. Heterosexually bound phage clones were highly enriched from 3.31 (-4) in the first round to 7.65 (-2) in the fourth round. Polyclonal phage Elisa assay also showed a clear enrichment effect. Eighty clones were randomly selected and infected with M13K07, and scFv was displayed on the surface of phage particles. Anti-A1 1-42 peptide scFv was screened by ELISA. Phage cloning. The results showed that 7 positive clones were obtained, of which 4 clones were 5 times higher than those of the negative control and 3 clones were 2.5-3 times higher than those of the negative control. The relative molecular weight of the target protein was 33 kD, mainly concentrated in the whole cell extract, accounting for about 60% of the total bacterial protein. The A value in the supernatant and the whole bacterial protein detected by ELISA was 5 times higher than that in the negative control. The antibody also binds to A-beta 1-40, but does not cross-react with BSA. It shows that scFv antibody is specific to A-beta, and the binding site is mainly at the N-terminal of A-beta. The biological activity of the expressed antibody shows that it can bind to the antigen A-beta fibers in amyloid plaques of human brain lesions, which proves that the expressed scFv antibody is specific to A-beta. Fv antibody can be applied to biological effect test in vivo. The sequence of scFv gene from A10 and F2 clones was sequenced and analyzed by blast. The DNA sequences of VH and VL were determined respectively. The deduced amino acid sequences had typical antibody variable region structure. The sequences of the two clones were identical, which indicated that the two clones might originate from the same strain. Primordial bacteriophage.
Two, construction of recombinant human anti A beta antibody and eukaryotic expression.
Firstly, the mature signal peptide genes of VH and VL genes were obtained by NCBI blast, and the signal peptide genes were synthesized. Specific primers were designed to amplify the CH, CL, FC genes of A10 cloned VH, VL, scFv genes and human IgG, respectively. The recombinant PCR (overlap PCR) method was used to recombine the full-antibody heavy chain of IgG. The whole IgG antibody light chain gene (L signal peptide gene + VL gene + CL gene) and the whole antibody single chain gene (H signal peptide gene + scFv gene + FC segment gene) were cloned into the eukaryotic expression vector pcDNA3.1 respectively. The results of bacterial fluid PCR, double enzyme digestion and DNA sequencing showed that the recombinant three gene fragments were identified. The gene sequence was exactly the same as expected, and the reading frame and the direction of insertion into eukaryotic expression vector were completely correct. The results of ELISA showed no positive reaction between the supernatant of the two transfected cells and A-beta 1-42. Further work still needs to be done to optimize the cell expression conditions.
At present, there are several reports of screening human anti-A beta antibodies at home and abroad, but they are still in the stage of laboratory research, and there are no reports of full antibodies or clinical trials. The patents have been declared, and the construction of the eukaryotic expression system of the whole antibody has been completed. The subsequent optimization of the expression system is under way. The results of this study will be helpful for further research on immunotherapy and clinical application of AD.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392
本文編號：2237946
[Abstract]:Alzheimer disease (AD) is a degenerative disease of the central nervous system characterized by progressive memory impairment and mental decline in the elderly. Senile plaque (SP), neurofibrillary tangles (NFT) and neurosynapse loss are the main pathological features of AD. Sign. The main component of senile plaque is the fibers formed by the aggregation of beta-amyloid peptide (A beta), which is thought to be the main pathogenic substance leading to neuronal damage and cognitive decline. Anti-A-beta polypeptide antibody has been proved to be effective in clearing the plaque of A-beta fibers in the brain of mice to prevent and treat AD.
In this study, we screened the variable region (scFv) gene of single-chain antibody against A beta from human phage single-chain antibody library and constructed the eukaryotic expression system of human anti-A beta whole antibody by genetic engineering. Soluble expression and detection of antibody binding activity and biological activity; Secondly, recombinant IgG antibody light and heavy chain gene and antibody single chain gene, construct eukaryotic expression system, and preliminary study of eukaryotic expression.
I. screening, prokaryotic expression and identification of human A scFv gene
Four rounds of enrichment and screening of the antibody library against A-beta-1-42 polypeptide were carried out. E.coli TG1 transformed strain was infected with auxiliary phage M13K07 and phage-like antibody library was displayed. The phage library was added to 96-well plate coated with A-beta-42 polypeptide and washed after incubation for a period of time. The scFv phage clone which could specifically bind to the antigen was retained. After neutralization with Tris-HCl at pH 8.0, E.coli TG1, which was in logarithmic growth phase, was infected to amplify antigen-specific phage clones. Thus, after four rounds of "adsorption-elution-amplification" enrichment, the antibody library was specific to the peptide A-beta 1-42. Heterosexually bound phage clones were highly enriched from 3.31 (-4) in the first round to 7.65 (-2) in the fourth round. Polyclonal phage Elisa assay also showed a clear enrichment effect. Eighty clones were randomly selected and infected with M13K07, and scFv was displayed on the surface of phage particles. Anti-A1 1-42 peptide scFv was screened by ELISA. Phage cloning. The results showed that 7 positive clones were obtained, of which 4 clones were 5 times higher than those of the negative control and 3 clones were 2.5-3 times higher than those of the negative control. The relative molecular weight of the target protein was 33 kD, mainly concentrated in the whole cell extract, accounting for about 60% of the total bacterial protein. The A value in the supernatant and the whole bacterial protein detected by ELISA was 5 times higher than that in the negative control. The antibody also binds to A-beta 1-40, but does not cross-react with BSA. It shows that scFv antibody is specific to A-beta, and the binding site is mainly at the N-terminal of A-beta. The biological activity of the expressed antibody shows that it can bind to the antigen A-beta fibers in amyloid plaques of human brain lesions, which proves that the expressed scFv antibody is specific to A-beta. Fv antibody can be applied to biological effect test in vivo. The sequence of scFv gene from A10 and F2 clones was sequenced and analyzed by blast. The DNA sequences of VH and VL were determined respectively. The deduced amino acid sequences had typical antibody variable region structure. The sequences of the two clones were identical, which indicated that the two clones might originate from the same strain. Primordial bacteriophage.
Two, construction of recombinant human anti A beta antibody and eukaryotic expression.
Firstly, the mature signal peptide genes of VH and VL genes were obtained by NCBI blast, and the signal peptide genes were synthesized. Specific primers were designed to amplify the CH, CL, FC genes of A10 cloned VH, VL, scFv genes and human IgG, respectively. The recombinant PCR (overlap PCR) method was used to recombine the full-antibody heavy chain of IgG. The whole IgG antibody light chain gene (L signal peptide gene + VL gene + CL gene) and the whole antibody single chain gene (H signal peptide gene + scFv gene + FC segment gene) were cloned into the eukaryotic expression vector pcDNA3.1 respectively. The results of bacterial fluid PCR, double enzyme digestion and DNA sequencing showed that the recombinant three gene fragments were identified. The gene sequence was exactly the same as expected, and the reading frame and the direction of insertion into eukaryotic expression vector were completely correct. The results of ELISA showed no positive reaction between the supernatant of the two transfected cells and A-beta 1-42. Further work still needs to be done to optimize the cell expression conditions.
At present, there are several reports of screening human anti-A beta antibodies at home and abroad, but they are still in the stage of laboratory research, and there are no reports of full antibodies or clinical trials. The patents have been declared, and the construction of the eukaryotic expression system of the whole antibody has been completed. The subsequent optimization of the expression system is under way. The results of this study will be helpful for further research on immunotherapy and clinical application of AD.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392
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