發(fā)布時(shí)間：2018-05-03 01:18

  本文選題：大腸癌 + 嵌合抗體��； 參考：《第四軍醫(yī)大學(xué)》2008年博士論文

【摘要】： 本實(shí)驗(yàn)以HAb18G/CD147- HAb18抗原抗體系統(tǒng)為基礎(chǔ),構(gòu)建并篩選了用于全人抗體表達(dá)的高效真核載體;之后,克隆了抗人大腸癌抗體CAb1輕、重鏈可變區(qū)基因VH和VL,通過(guò)制備復(fù)性的嵌合cFab對(duì)獲得的基因的可靠性和準(zhǔn)確性進(jìn)行驗(yàn)證;進(jìn)而以獲得的CAb1的VH和VL為基礎(chǔ),對(duì)其進(jìn)行人源化表面重塑抗體rCAb1設(shè)計(jì);最終,選用鑒定的抗體真核表達(dá)載體,實(shí)現(xiàn)了重組rCAb1在哺乳動(dòng)物細(xì)胞中的表達(dá)。 第一部分研究?jī)?nèi)容:高效全人抗體真核表達(dá)載體的構(gòu)建及篩選 目的:獲得具有自主產(chǎn)權(quán),且能表達(dá)全人抗體的高效真核表達(dá)載體 方法:按照標(biāo)準(zhǔn)DNA重組操作,構(gòu)建以下不同的真核表達(dá)載體:包括弱化多順?lè)醋觩IRES1-18L;pIRES2-18H;定點(diǎn)重組載體18-pDHL-FRT;雙載體系統(tǒng)18H-pDHA,18L-pCI;反向雙載體系統(tǒng)18H-PCI,18L-p105;多順?lè)醋颖磉_(dá)載體18H-L-pCI-FRT;弱化篩選標(biāo)記載體18H-L-pCII;以及pAH/pAG雙載體pAH4604-18,pAG4622-18,這些載體均含有單抗HAb18輕重鏈可變區(qū)基因和人IgG1κ的輕、重鏈恒定區(qū)基因。之后,將這些載體分別轉(zhuǎn)染COS-7細(xì)胞,斑點(diǎn)雜交檢測(cè)培養(yǎng)上清的情況,觀測(cè)是否有嵌合抗體cHAb18的表達(dá);用夾心ELISA法檢測(cè)重組抗體的表達(dá)量。隨后,選用部分構(gòu)建載體,用電穿孔穩(wěn)定轉(zhuǎn)染不同表型的CHO細(xì)胞,有限稀釋進(jìn)行陽(yáng)性克隆篩選,完成轉(zhuǎn)染細(xì)胞的篩選;RT-PCR檢測(cè)穩(wěn)定傳代的細(xì)胞系,觀察抗體基因是否丟失,評(píng)估轉(zhuǎn)染載體的細(xì)胞穩(wěn)定性。進(jìn)行陽(yáng)性克隆的擴(kuò)大培養(yǎng),表達(dá)產(chǎn)物的純化;SDS-PAGE凝膠電泳及其Western blot檢測(cè)抗體的表達(dá)情況;選用細(xì)胞免疫熒光染色以及流式細(xì)胞檢測(cè)表達(dá)產(chǎn)物的特異性。 結(jié)果:構(gòu)建了含有抗體基因的不同的真核載體,通過(guò)必要的PCR或酶切鑒定插入序列,均獲得了和理論上大小一致的片段,提示所有的載體均成功構(gòu)建。將其全部瞬時(shí)轉(zhuǎn)染COS-7細(xì)胞,斑點(diǎn)雜交結(jié)果顯示,與未轉(zhuǎn)化的細(xì)胞相比,除載體18H-L-pCII與雙載體pAH/pAG沒(méi)有檢測(cè)到表達(dá)的IgG外,其余的載體均能檢測(cè)到抗體的表達(dá);而用抗原HAb18G/CD147胞外區(qū)進(jìn)行的夾心ELISA結(jié)果證實(shí):所有重組表達(dá)載體轉(zhuǎn)化后均能成功檢測(cè)到嵌合抗體cHAb18的表達(dá),所有的載體均在轉(zhuǎn)染48h后表達(dá)量達(dá)到較高水平,120 h后表達(dá)量達(dá)到最高;其中雙載體pIRES1-18L/pIRES2-18H和pDHL-18FRT的量約達(dá)到1.4 mg/L。進(jìn)一步選用三種不同的載體pIRES1-18L/pIRES2-18H,pDHL-18FRT和18H-L-pCII進(jìn)行穩(wěn)定轉(zhuǎn)染不同表型的CHO細(xì)胞。夾心ELISA結(jié)果顯示上述不同的三種載體均較未轉(zhuǎn)化的細(xì)胞有人IgG的表達(dá),其中雙載體系統(tǒng)pIRES1-18L/pIRES2-18H的表達(dá)量約介于0.3-16mg/L;載體pDHL-18FRT的表達(dá)量可達(dá)到10mg/L;但是載體18H-L-pCII的抗體表達(dá)量,在進(jìn)行克隆化并加壓篩選的情況下卻沒(méi)有太多的變化,表達(dá)量均少于1.5 mg/L。對(duì)pIRES1-18L/pIRES2-18H轉(zhuǎn)染后的細(xì)胞株1F6,穩(wěn)定傳代培養(yǎng)30代,RT-PCR仍能檢測(cè)到抗體基因的表達(dá);進(jìn)一步擴(kuò)大培養(yǎng),親合層析柱從約500ml的培養(yǎng)上清中純化,獲得約了7.5mg的表達(dá)產(chǎn)物,產(chǎn)物的純度較高。SDS-PAGE電泳和Western blot檢測(cè)結(jié)果:目的蛋白分子量約為150kDa,還原后為兩條帶,分子量約為50kDa和25kDa,符合人IgG抗體的特征,且可以和羊抗人IgG結(jié)合。免疫熒光結(jié)果顯示表達(dá)純化的抗體可特異結(jié)合表達(dá)有HAb18G/CD147的細(xì)胞。 結(jié)論:通過(guò)對(duì)載體的優(yōu)化設(shè)計(jì)和改造,獲得了能用于全人抗體表達(dá)的高效真核載體;其中以弱化多順?lè)醋觩IRES1-18L; pIRES2-18H以及定點(diǎn)重組載體18-pDHL-FRT表達(dá)量較高,均可進(jìn)一步用于其他抗體的表達(dá)。成功的篩選了抗HAb18G/CD147的嵌合IgG抗體cHAb18的穩(wěn)定細(xì)胞株,并在哺乳動(dòng)物細(xì)胞中獲得高效表達(dá),表達(dá)產(chǎn)物保持了良好的特異性和親合力。純化了重組表達(dá)的目標(biāo)全長(zhǎng)IgG產(chǎn)物即人鼠嵌合抗體cHAb18,從而也進(jìn)一步驗(yàn)證了所獲得的表達(dá)載體是正確的且能用于表達(dá)全長(zhǎng)人IgG。 第二部分研究?jī)?nèi)容:抗人大腸癌單抗CAb1輕、重鏈基因克隆與鑒定 目的:獲得單抗CAb1的輕、重鏈可變區(qū)基因,及其嵌合Fd或嵌合輕鏈基因,制備相應(yīng)的小分子抗體cFab對(duì)所獲得的基因進(jìn)行鑒定。 方法:首先,從雜交瘤細(xì)胞CAb1提取總RNA,設(shè)計(jì)并選用合成引物,通過(guò)反轉(zhuǎn)錄PCR擴(kuò)增單抗CAb1 Fd和輕鏈全長(zhǎng)基因,連接人T載體后進(jìn)行序列測(cè)定和分析;然后用同源比較對(duì)單抗CAb1完整Fd的擴(kuò)增,獲得含有全長(zhǎng)的Fd和輕鏈基因的載體pMD18-T/Fd和pMD18-T/L;分別以pMD18-T/Fd和pMD18-T/L為模版,用對(duì)應(yīng)的引物B4、B4for和A8、A8for擴(kuò)增MAbCAb1重、輕鏈可變區(qū)基因VH和VL,分別插入含有人CH1和CL的表達(dá)載體pComb3C后獲得載體pComb3C/cFab;以載體pComb3C/cFab為模板,再次采用相應(yīng)的引物擴(kuò)增嵌合Fd或嵌合輕鏈基因,用相應(yīng)的限制性內(nèi)切酶分別消化質(zhì)粒pET32a(+)及純化的PCR產(chǎn)物,經(jīng)過(guò)連接轉(zhuǎn)化鑒定獲得載體pET-CAbH和pET-CAbL。誘導(dǎo)表達(dá)含有重組表達(dá)質(zhì)粒的菌株,分別表達(dá)嵌合cFd和cL并進(jìn)行純化。將其分別溶解后,按絕對(duì)量等摩爾量比混合,用梯度透析的方法進(jìn)行復(fù)性。SDS-PAGE和Westernblot對(duì)嵌合Fab進(jìn)行檢測(cè),觀察產(chǎn)物的復(fù)性情況;用ProteinG的柱子純化產(chǎn)物后,選用間接ELISA,免疫熒光染色,流式細(xì)胞術(shù)檢測(cè)復(fù)性產(chǎn)物的靶抗原結(jié)合活性,最后采用競(jìng)爭(zhēng)性ELISA檢測(cè)該小分子抗體和鼠源單抗CAb1是否競(jìng)爭(zhēng)同一個(gè)抗原表位。 結(jié)果:從1×107的雜交瘤細(xì)胞CAb-1細(xì)胞中獲得總RNA量約62.5μg。甲醛變性電泳顯示總RNA比較完整,能夠準(zhǔn)確清晰的觀察到5S,18S和28S的銳利條帶。獲得mAbCAb1基因序列,VH基因長(zhǎng)351bp,編碼117個(gè)氨基酸;VL基因長(zhǎng)336bp,編碼112個(gè)氨基酸;CAb-1CH1屬于IgG1,CL屬于κ型。優(yōu)化其mRNA的二級(jí)結(jié)構(gòu)自由能,構(gòu)建了非融合表達(dá)載體pET-CAbH,pET-CAbL,成功實(shí)現(xiàn)了抗體的嵌合Fd或嵌合輕鏈在大腸桿菌中的高效表達(dá),蛋白表達(dá)量在30°C誘導(dǎo)6h后分別達(dá)到菌體總蛋白的23.6%和29.2%。通過(guò)體外復(fù)性,SDS-PAGE結(jié)果顯示在起始總蛋白濃度100μg/ml時(shí),復(fù)性的cFab的回收率高達(dá)70.2%。免疫熒光和FACS的結(jié)果顯示復(fù)性的cFab能夠比較特異的結(jié)合到大腸癌細(xì)胞系SW480和Hce-8693,但是卻不和正常的細(xì)胞結(jié)合。競(jìng)爭(zhēng)ELISA檢測(cè)顯示復(fù)性的cFab和親本CAb-1抗體片段F(ab')2互相競(jìng)爭(zhēng)相同表位。結(jié)論:獲得了雜交瘤細(xì)胞CAb1的輕重鏈可變區(qū)基因,所設(shè)計(jì)的引物對(duì)鼠IgG1類抗體基因的擴(kuò)增是有效的。表達(dá)并制備了嵌合cFd和cL,優(yōu)化mRNA的二級(jí)結(jié)構(gòu)自由能對(duì)蛋白的非融合表達(dá)具有重要作用。實(shí)現(xiàn)體外復(fù)性制備小分子cFab,該分子能特異的結(jié)合大腸癌細(xì)胞,所獲得的分子具有結(jié)合活性;復(fù)性的cFab能和親本的CAb-1相互競(jìng)爭(zhēng),提示它們識(shí)別的抗原表位一致;由于制備的cFab抗原有結(jié)合活性,進(jìn)一步提示所獲得雜交瘤細(xì)胞CAb1基因是正確和可靠的,同時(shí)也印證了該方案對(duì)鑒定所克隆的CAb1可變區(qū)基因的有效性。 第三部分研究?jī)?nèi)容:抗人大腸癌表面重塑抗體rCAb1表達(dá)和純化目的:用構(gòu)建的真核表達(dá)載體制備抗人大腸癌表面重塑抗體rCAb1,對(duì)表達(dá)的抗體進(jìn)行初步純化和鑒定 方法:首先通過(guò)對(duì)大腸癌單抗CAb1的抗體可變區(qū)序列分析,并同Genbank的nr庫(kù)做BlastP,從中抽取所有抗體可變區(qū)氨基酸序列信息構(gòu)建本地抗體結(jié)構(gòu)數(shù)據(jù)庫(kù)。進(jìn)而將抗體種屬來(lái)源結(jié)合輕、重鏈類型將抗體序列分為四類,其中抗體序列來(lái)源分別選擇Homo sapiens和Mus musculus兩類。對(duì)相似性得分最高的人源、鼠源抗體可變區(qū)序列各200條進(jìn)行統(tǒng)計(jì),獲得單抗CAb1的差異殘基和異常殘基;之后通過(guò)模建抗體可變區(qū)的精確三維模型,獲得CAb1的VH、VL分子內(nèi)和分子間氫鍵相互作用,最終確定進(jìn)行人源化改造的候選突變位點(diǎn);依據(jù)確定的候選位點(diǎn),對(duì)突變后的抗體人源化可變區(qū)序列用重疊PCR的方法合成,分別與T載體(pMD18-T)連接構(gòu)建成克隆載體pMD18-T/VH和pMD18-T/VL進(jìn)行序列測(cè)定。利用上述的可變區(qū)序列,分別構(gòu)建輕鏈表達(dá)載體pIRES1-CAbL和重鏈表達(dá)載體的pIRES2-CAbH(弱化多順?lè)醋酉到y(tǒng)),用PCR和相應(yīng)的酶切鑒定。將獲得的雙載體轉(zhuǎn)染COS-7細(xì)胞進(jìn)行瞬時(shí)表達(dá),夾心ELISA法檢測(cè)抗體的表達(dá)情況。最后,將這兩個(gè)載體共轉(zhuǎn)CHO-dhfr-細(xì)胞,夾心ELISA法檢測(cè)產(chǎn)物的表達(dá)情況;用有限稀釋法進(jìn)行陽(yáng)性克隆的篩選,擴(kuò)大培養(yǎng)后進(jìn)行抗體表達(dá)產(chǎn)物的純化,純化樣品進(jìn)行SDS-PAGE凝膠電泳分析和Western blot檢測(cè)。細(xì)胞免疫熒光染色以及流式細(xì)胞檢測(cè)觀察純化產(chǎn)物對(duì)大腸癌細(xì)胞的結(jié)合活性。最后選用競(jìng)爭(zhēng)性ELISA檢測(cè)該rCAb1是否與親本鼠CAb1競(jìng)爭(zhēng)同一表位,同時(shí)根據(jù)50%抑制率時(shí)的人源抗體與親本鼠抗體濃度的比值,估計(jì)該人源化IgG的相對(duì)親和力。 結(jié)果:按照Kabat、Abm、Chothia和Contact的規(guī)則標(biāo)出了大腸癌單抗CAb1的不同的CDR,SDR區(qū);經(jīng)過(guò)篩選的抗體序列經(jīng)過(guò)分析,獲得了已有抗體分子不同位點(diǎn)上氨基酸種類和百分比,將該結(jié)果和單抗CAb1的可變區(qū)氨基酸比對(duì)后,獲得了該序列的差異殘基和異常殘基;結(jié)合三維重建模型,獲得了CAb1 VH、VL分子內(nèi)和分子間氫鍵相互作用和氨基酸表面可及性,并最終確定了初步進(jìn)行人源化改造的候選突變位點(diǎn)即重鏈的T018S,N086S和輕鏈的Q018P。重疊PCR合成突變的基因后,分別與T載體連接,獲得了克隆載體pMD18-T/VH和pMD18-T/VL,測(cè)序結(jié)果顯示可變區(qū)基因成功合成。構(gòu)建的輕鏈表達(dá)載體pIRES1-CAbL和重鏈表達(dá)載體的pIRES2-CAbH經(jīng)轉(zhuǎn)染COS-7細(xì)胞后,夾心ELISA證明存在人IgG的表達(dá);將這兩個(gè)載體共轉(zhuǎn)CHO-dhfr-細(xì)胞,經(jīng)過(guò)克隆篩選,表達(dá)產(chǎn)物的純化,SDS-PAGE電泳和Western blot檢測(cè)結(jié)果表明:在非還原狀態(tài)下,目標(biāo)蛋白的分子量約為150 kDa;還原后為兩條帶,分子量分別約為52kDa和27kDa。該產(chǎn)物可特異的和大腸癌細(xì)胞結(jié)合。競(jìng)爭(zhēng)性ELISA檢測(cè)該產(chǎn)物即rCAb1可與親本鼠CAb1競(jìng)爭(zhēng)同一表位,且親和力約為親本鼠抗體的55%。 結(jié)論:通過(guò)對(duì)大腸癌單抗CAb1的抗體可變區(qū)序列分析,獲得了對(duì)大腸癌單抗CAb1進(jìn)行表面重塑改造的候選突變位點(diǎn);成功的用Over-lapping PCR的方法合成了突變的CAb1輕、重鏈可變區(qū)基因;用弱化篩選標(biāo)記的雙載體pIRES1-CAbL和pIRES2-CAbH制備了表面重塑抗體rCAb1;所制備的抗體能特異的結(jié)合大腸癌細(xì)胞;保持了和親本抗體相似的結(jié)合活性和特異性。
[Abstract]:On the basis of the HAb18G/CD147- HAb18 antigen antibody system, the high effective eukaryotic vector used for the expression of human antibody was constructed and screened. After that, the anti colon cancer antibody CAb1 light, the heavy chain variable region gene VH and VL were cloned, and the reliability and accuracy of the genes obtained by the preparation of complex chimeric cFab were verified, and then the results were obtained. Based on the VH and VL of CAb1, the human derived surface remodeling antibody rCAb1 was designed. Finally, the expressed eukaryotic expression vector was selected to realize the expression of recombinant rCAb1 in mammalian cells.
The first part is about the construction and screening of eukaryotic expression vector of high effective human antibody.
Objective: to obtain a high efficient eukaryotic expression vector with independent property rights and full human antibody expression.
Methods: according to the standard DNA recombination operation, the following different eukaryotic expression vectors are constructed, including the weakening of the polycis pIRES1-18L, pIRES2-18H, the fixed-point recombinant vector 18-pDHL-FRT, the dual carrier system 18H-pDHA, 18L-pCI, the reverse dual carrier system 18H-PCI, 18L-p105, the polycis anti subexpression vector 18H-L-pCI-FRT, and the weakening screening marker carrier 18H-L-pCII; And pAH/pAG dual carrier pAH4604-18, pAG4622-18, these carriers all contain the light heavy chain gene of mAb HAb18 and the light, heavy chain constant region gene of human IgG1 kappa. After that, these vectors are transfected to COS-7 cells respectively, and dot blot hybridization is used to detect the culture of supernatant, and the expression of the inlay antibody cHAb18 is observed, and the sandwich ELISA method is used to detect the recombination resistance. Then, the CHO cells with different phenotypes were transfected steadily with electroporation, and the positive clones were screened with limited dilution to screen the positive clones, and the transfected cells were screened. RT-PCR was used to detect the stable cell lines, to observe whether the antibody genes were lost, and to evaluate the cell stability of the transfer carriers. The expression of the product was purified, the expression of the antibody was detected by SDS-PAGE gel electrophoresis and its Western blot, and the specificity of the expression products was detected by cell immunofluorescence staining and flow cytometry.
Results: the different eukaryotic vectors containing the antibody genes were constructed, and the inserted sequences were identified by the necessary PCR or enzyme digestion. All the vectors were successfully constructed. All the vectors were successfully constructed. All the transfected COS-7 cells were transiently transfected. The dot blot hybridization showed that the carrier 18H-L-pCII was compared with the Unconverted cells. The expression of the antibody was detected by the other carriers without detection of the expression of IgG, while the ELISA results of the sandwich HAb18G/CD147 in the extracellular domain of the antigen HAb18G/CD147 confirmed that all the recombinant expression vectors could successfully detect the expression of the chimeric antibody cHAb18 after the transformation of all the recombinant vectors, and all the carriers were expressed in high water after transfection of 48h. After 120 h, the expression reached the highest; the amount of pIRES1-18L/pIRES2-18H and pDHL-18FRT reached about 1.4 mg/L., and three different carriers, pIRES1-18L/pIRES2-18H, pDHL-18FRT and 18H-L-pCII were used to transfect the CHO cells with different phenotypes. The sandwich ELISA results showed that all the three different carriers were not transformed. The expression of IgG, the expression of pIRES1-18L/pIRES2-18H in the double carrier system is about 0.3-16mg/L, and the expression of carrier pDHL-18FRT can reach 10mg/L, but the expression of the antibody of carrier 18H-L-pCII is not much changed under the condition of cloning and pressure screening, and the expression amount is less than 1.5 mg/L. to pIRES1-18L/pIRE. The transfected cell line, 1F6, was successfully cultured for 30 generations, and the expression of the antibody gene was still detected by RT-PCR. Further expanded culture, the affinity chromatography column was purified from the culture supernatant of about 500ml, and the expression product of 7.5mg was obtained. The purity of the product was high.SDS-PAGE electrophoresis and Western blot detection results: the molecular weight of the target protein was about 150k Da, after reduction, is two bands, with a molecular weight of about 50kDa and 25kDa, which conforms to the characteristics of human IgG antibody and can be combined with the anti human IgG of sheep. The immunofluorescence results show that the purified antibody can specifically express HAb18G/CD147 cells.
Conclusion: by optimizing the design and modification of the carrier, a high effective eukaryotic vector can be obtained for the expression of the whole human antibody. The high expression of the weak polycis pIRES1-18L, pIRES2-18H and the fixed-point recombinant vector 18-pDHL-FRT can be further used for the expression of other antibodies. The anti HAb18G/CD147 chimeric IgG antibody is successfully screened. The stable cell line of cHAb18 is highly expressed in mammalian cells, and the expression product maintains good specificity and affinity. The full IgG product of the recombinant expression is purified, that is, human chimeric antibody cHAb18, which further verifies that the obtained expression vector is correct and can be used to express the full length human IgG..
The second part is about cloning and identification of light and heavy chain genes of monoclonal antibody CAb1 against human colorectal cancer.
Objective: to obtain the light and heavy chain variable region genes of the monoclonal antibody CAb1 and their chimeric Fd or chimeric light chain genes, and to prepare the corresponding small molecular antibody cFab to identify the obtained genes.
Methods: first, the total RNA was extracted from the hybridoma cell CAb1, and the synthetic primers were designed and selected. The monoclonal antibody CAb1 Fd and the light chain full length gene were amplified by reverse transcription PCR, and the T vector was sequenced and analyzed. Then the full length Fd and light chain gene carrier pMD18-T/Fd and pMD1 were obtained by the homologous comparison of the complete Fd of the monoclonal antibody CAb1. 8-T/L, pMD18-T/Fd and pMD18-T/L were used as templates to amplify MAbCAb1 weight, VH and VL with corresponding primers B4, B4for and A8, A8for in light chain variable region gene, and the vector containing the expression vector containing human CH1 and CL were inserted respectively, and the vector was used as the template to amplify the chimeric or chimeric light chain base with the corresponding primers. The plasmid pET32a (+) and purified PCR products were digested with the corresponding restriction endonuclease, and the recombinant plasmid containing the recombinant expression plasmid was induced by the connection transformation, and the recombinant plasmid containing the recombinant expression plasmid was expressed by pET-CAbH and pET-CAbL., and the chimeric cFd and cL were expressed and purified respectively. The compound.SDS-PAGE and Westernblot were used to detect the chimeric Fab and observe the refolding of the products. After purifying the products by the ProteinG column, indirect ELISA, immunofluorescence staining, and flow cytometry were used to detect the target antigen binding activity of the complex products. Finally, the competitive ELISA was used to detect the small molecular antibody and the rat monoclonal antibody CAb1. Whether the same antigen epitopes are competing.
Results: the total RNA was obtained from 1 * 107 hybridoma cells CAb-1 cells, and the total RNA was about 62.5 Mu g. formaldehyde denaturation. The total RNA was more complete, and the sharp bands of 5S, 18S and 28S could be accurately and clearly observed. The mAbCAb1 gene sequence was obtained, the VH gene was long 351bp, 117 amino acids were encoded, VL gene long 336bp and 112 amino acids were encoded. L belongs to kappa type. Optimize the two level structural free energy of its mRNA, construct a non fusion expression vector pET-CAbH, pET-CAbL, successfully realized the high expression of the chimeric Fd or chimeric light chain in Escherichia coli. The protein expression reached 23.6% of the total protein of the bacterial body after the induction of 6h by 30 degree C and the expression of the protein was refolding in vitro, and the SDS-PAGE result was shown. When the initial total protein concentration was 100 g/ml, the recovery of the complex cFab was up to 70.2%. immunofluorescence and FACS. The complex cFab could be more specifically binding to the colorectal cancer cell line SW480 and Hce-8693, but did not combine with the normal cells. Competitive ELISA detection showed that the refolding cFab and the parent CAb-1 antibody fragment F (ab') 2 each other. Conclusion: the variable region gene of hybridoma cell CAb1 was obtained. The primers designed to amplify the IgG1 antibody gene of rat were effective. The expression and preparation of chimeric cFd and cL were expressed and prepared, and the two level structural free energy of mRNA was important for the non fusion expression of the protein, and the small molecule cFab was prepared in vitro. The molecules can specifically bind colorectal cancer cells, the molecules obtained by the molecules have binding activity, and the complex cFab can compete with the parent CAb-1, suggesting that the antigen epitopes identified by them are the same. Because of the binding activity of the prepared cFab antigen, it is suggested that the hybridoma CAb1 gene is correct and reliable, and is also proved to be the same. The protocol was applied to identify the CAb1 variable region gene cloned.
The third part of the study: anti human colorectal cancer surface remodeling antibody rCAb1 expression and purification purpose: to prepare the anti colon cancer surface remodeling antibody rCAb1 with the constructed eukaryotic expression vector, and to purify and identify the expressed antibody.
Methods: first, by analyzing the antibody variable region sequence of the monoclonal antibody CAb1 of colorectal cancer, and using the NR Library of Genbank as BlastP, the local antibody structure database was constructed from all the antibody variable region amino acid sequence information. Then the antibody species belonged to the light, heavy chain type and the anti body sequence was divided into four classes, in which the antibody sequence sources were respectively derived. Homo sapiens and Mus musculus two were selected. The difference residues and abnormal residues of the mAb CAb1 were obtained by the 200 strips of the human source of the mouse antibody variable region, and then the VH of CAb1, the intramolecular and intermolecular hydrogen bond interaction of the VL were obtained by the exact three-dimensional model of the variable region of the model antibody. Candidate mutant loci for the transformation of pedestrians; based on the identified candidate loci, the mutant antibody humanized variable region sequence was synthesized by overlapping PCR, and the T vector (pMD18-T) was constructed into a clone vector, pMD18-T/VH and pMD18-T/VL, respectively. The light chain expression vector pIR was constructed by using the previous variable region sequence. ES1-CAbL and heavy chain expression vector pIRES2-CAbH (weakening polycis anti subsystem), identified by PCR and corresponding enzyme digestion. The transfected COS-7 cells were transiently expressed and the ELISA method was used to detect the expression of antibodies. Finally, the two vectors were converted to CHO-dhfr- fine cell and the ELISA method of sandwich was used to detect the expression of the products. The dilution method was used to screen the positive clones, the antibody expression products were purified, the purified samples were analyzed by SDS-PAGE gel electrophoresis and Western blot detection. Cell immunofluorescence staining and flow cytometry were used to observe the binding activity of the purified products to colorectal cancer cells. Finally, competitive ELISA was selected to detect the rCAb1. The relative affinity of the human IgG was estimated at the same epitopes with the parent mouse CAb1, and the ratio of the human antibody to the parent mouse antibody concentration at the 50% inhibition rate was estimated.
Results: according to the rules of Kabat, Abm, Chothia and Contact, the different CDR, SDR region of the large intestine cancer McAb CAb1 was identified. The selected antibody sequence was analyzed to obtain the amino acid species and percentage of the existing antibody molecules, and the result was compared with the variable region amino acid of the monoclonal antibody CAb1, and the difference residues of the sequence were obtained. CAb1 VH, VL intermolecular and intermolecular hydrogen bond interaction and the accessibility of amino acid surface were obtained by the three-dimensional reconstruction model. Finally, the candidate mutation sites for the preliminary humanized transformation, namely, the heavy chain T018S, N086S and the Q018P. overlap of the light chain, were determined to be connected with T vectors respectively. PMD18-T/VH and pMD18-T/VL were cloned, and the sequencing results showed that the variable region gene was successfully synthesized. The constructed light chain expression vector pIRES1-CAbL and the pIRES2-CAbH of heavy chain expression vector were transfected to COS-7 cells, and the sandwich ELISA showed the expression of human IgG; the two vectors were converted to CHO-dhfr- cells, and the expression products were purified by cloning and the purity of the products was pure. The results of SDS-PAGE electrophoresis and Western blot detection showed that the molecular weight of the target protein was about 150 kDa in the non reductive state; two bands were reduced after reduction and the molecular weight was about 52kDa and 27kDa., respectively. The competitive ELISA detected the product that rCAb1 could compete with the parent rat CAb1 in the same epitope and affinity. 55%., which is about the antibody of the parent rat
Conclusion: the monoclonal antibody CAb1 of colorectal cancer was obtained by analyzing the variable region sequence of monoclonal antibody CAb1 of colorectal cancer.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392;Q78

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本文編號(hào)：1836333