本文選題：膠原酶　切入點：單域　出處：《中國協(xié)和醫(yī)科大學》2009年博士論文　論文類型：學位論文

【摘要】： 力達霉素(lidamycin,LDM),原名C-1027,是一種新型的烯二炔類抗腫瘤抗生素,由球孢鏈霉菌C-1027(Streptomyces globisporus C-1027)產生。其抗腫瘤活性比臨床常用的阿霉素強10,000倍,已經進入臨床Ⅱ期試驗。力達霉素由一個酸性輔基蛋白CagA(C-1027 apoprotein、C-1027AG,也寫作LDP)和一個烯二炔結構的發(fā)色團(chromophore,也寫作LDC)非共價結合組成,發(fā)色團為活性組分,輔基蛋白保護和運載發(fā)色團。由于力達霉素自身具有輔基蛋白,可以將其與抗腫瘤抗體偶聯(lián)成為融合蛋白,改造為靶向特異腫瘤的載體,構建導向性力達霉素。Ⅳ型膠原酶降解細胞基底膜中的Ⅳ型膠原,破壞基底膜及細胞外基質的完整性,引起腫瘤細胞的侵襲與轉移,Ⅳ型膠原酶已經成為抗腫瘤研究的靶標�？耿粜湍z原酶抗體可以抑制Ⅳ型膠原酶的活性并可作為導向藥物的載體。 本工作根據已知的抗Ⅳ型膠原酶單鏈抗體基因序列,設計合成了具有鏈霉菌偏好密碼子的抗Ⅳ型膠原酶單域抗體V_H(抗Ⅳ型膠原酶抗體重鏈可變區(qū))基因,構建了含有輔基蛋白與抗Ⅳ型膠原酶單域抗體融合蛋白基因cagA-V_H和用于篩選的阿普霉素抗性基因aac(3)Ⅳ的重組質粒pBSH,用接合轉移的方法導入力達霉素產生菌S.globisporus C-1027,通過同源重組雙交換,以cagA-V_H -aac(3)Ⅳ取代力達霉素生物合成基因簇中原有的輔基蛋白基因cagA,得到基因工程菌株S.globisporus C-1027 V_H,以獲得Ⅳ型膠原酶靶向的力達霉素。對基因工程菌株進行發(fā)酵,以枯草桿菌(Bacillus subtilis)為檢定菌,結果表明基因工程菌株發(fā)酵液具有一定的抑菌活性,但與原產生菌相比活性較低。經Western blot分析在胞內能檢測到融合蛋白和降解的輔基蛋白,而胞外僅檢測到降解的輔基蛋白條帶。ELISA檢測到融合蛋白的免疫活性。同時構建了含有融合蛋白基因cagA-V_H但不含抗性基因的重組質粒pBS03H,通過同源重組雙交換,以cagA-V_H基因取代cagA基因,得到僅融合了抗Ⅳ型膠原酶單域抗體基因的基因工程菌株S.globisporus C-1027NV_H。同樣進行了發(fā)酵、抑菌活性、SDS-PAGE和Western blot的檢測,結果與S.globisporus C-1027 V_H基本相同。 為了增加融合蛋白的表達量,獲得高活性的導向性力達霉素,本工作進一步構建了輔基蛋白阻斷株,并通過導入多拷貝的融合蛋白表達質粒的策略來提高融合蛋白的表達量。構建重組質粒pBSA,將質粒接合轉移至S.globisporus C-1027中,采用同源重組雙交換的方法將cagA基因阻斷。通過抗性篩選、PCR和Southern blot驗證,最終獲得輔基蛋白阻斷株,命名為S.globisporus AKO。用四種輔基蛋白表達質粒pKCTA900、pSETTA900、pLTA900和pLTA400分別導入在自身啟動子和/或強啟動子控制下的cagA基因對阻斷突變株S.globisporus AKO進行互補,得到相應的互補菌株。對阻斷株和互補菌株進行發(fā)酵,發(fā)酵液的抑菌活性和HPLC分析結果表明阻斷株完全喪失了力達霉素的產生能力,而互補菌株能不同程度地恢復產生力達霉素,其中S.globisporus AKO/pKCTA900基本恢復到野生株的水平。將質粒pKCA900、pSETA900、pLA900和pLA400接合轉移至野生株S.globisporusC-1027中構建了相應的輔基蛋白過表達菌株。抑菌活性和HPLC分析結果表明過量表達cagA基因可以提高力達霉素的產量。 構建輔基蛋白-單域抗體的融合蛋白表達質粒pKCTH2600、pSETTH2600分別導入輔基蛋白阻斷株中進行表達。重組菌株S.globisporus AKO/pKCTH2600發(fā)酵液的抑菌活性在發(fā)酵晚期與野生株相當,經Western blot分析在胞內、胞外均能檢測到融合蛋白的特異條帶。ELISA檢測到融合蛋白的免疫活性。結果表明融合蛋白在輔基蛋白阻斷株中以多拷貝質粒的形式獲得表達且表達量較基因工程菌株S.globisporus C-1027 V_H和S.globisporus C-1027 NV_H明顯提高。 綜上所述,本工作通過優(yōu)化融合基因的表達策略,提高了融合蛋白的表達水平,為獲得抗Ⅳ型膠原酶單域抗體V_H導向的力達霉素奠定了基礎,為進一步構建高表達的導向力達霉素重組菌株提供了新的技術平臺。
[Abstract]:Lidamycin (lidamycin, LDM), formerly known as C-1027, is a new type of graphene two acetylenic antitumor antibiotic by Streptomyces globisporus C-1027 (Streptomyces globisporus C-1027). The antitumor activity of doxorubicin than the clinical commonly used 10000 times stronger, has entered phase II clinical trials. Lidamycin consists of an acid apoprotein CagA (C-1027, apoprotein, C-1027AG, also called LDP and a two) ene alkynyl structure chromophore (chromophore, also called LDC) non covalent binding, chromophore as active component, apoprotein and carrier protection. Because of lidamycin chromophore has its own apoprotein, and can be anti tumor antibodies become fusion protein into specific tumor targeting vectors, construct the orientation of lidamycin. Type IV collagen type IV collagenase degradation cell basement membrane, destroy the integrity of basement membrane and extracellular matrix, caused by tumor Cell invasion and metastasis. Type IV collagenase has become a target of anti-tumor research. Anti type IV collagenase antibody can inhibit the activity of type IV collagenase and can be used as a carrier of targeting drug.
According to the work of anti type IV collagenase scFv gene sequence is known, anti type IV collagenase single domain antibody V_H with Streptomyces codon was designed and synthesized (heavy chain variable region of anti collagenase antibody) gene construct containing apoprotein and anti type IV collagenase single domain antibody fusion protein gene cagA-V_H and for screening of the Apramycin resistance gene AAC (3) recombinant plasmid pBSH IV, with the method of conjugation into lidamycin producing bacteria S.globisporus C-1027 by homologous recombination double exchange, cagA-V_H -aac (3) IV substituted lidamycin apoprotein biosynthesis gene cagA original gene cluster, by gene engineering strain S.globisporus C-1027 V_H, to obtain the type IV collagenase targeting lidamycin. On the fermentation of genetic engineering strain, Bacillus subtilis (Bacillus subtilis) as test strain, the results show that the genetic engineering Certain antibacterial activity with fermentation broth, but compared with the native bacterial activity is low. By Western blot analysis to the apoprotein and degradation of the fusion protein in the cell can be detected, and the extracellular only detected apoprotein bands.ELISA detected the immunological activity of the fusion protein degradation. At the same time construct containing fusion protein gene cagA-V_H without resistance gene recombinant plasmid pBS03H by homologous recombination double exchange, replacing the cagA gene with cagA-V_H gene, only the fusion gene engineering strain S.globisporus C-1027NV_H. anti type IV collagenase single domain antibody gene were also fermentation, antimicrobial activity, detection of SDS-PAGE and Western blot, and S.globisporus C-1027 V_H results basically the same.
In order to increase the expression of the fusion protein to obtain high activity oriented lidamycin, this work further constructs the apoprotein blocking line, and through the introduction of multiple copies of the fusion protein expression plasmid of the strategies to improve the expression level of the fusion protein. The recombinant plasmid pBSA plasmid conjugation to S.globisporus in C-1027 method the double exchange of homologous recombination cagA gene. By blocking resistance screening, PCR and Southern blot verification, finally obtain the apoprotein blocking strains, named S.globisporus AKO. with four apoprotein expression plasmid pKCTA900, pSETTA900, pLTA900 and pLTA400 respectively into its own promoter and / or promoter under the control of cagA the S.globisporus gene blocked mutant AKO were complementary, complementary strains. The corresponding fermentation to block strain and complementary strain analysis and antibacterial activity of HPLC fermentation liquid The results show that the AKO completely lost the ability to produce lidamycin, and complementary strains could be recovered at different levels of lidamycin, which S.globisporus AKO/pKCTA900 recovered to the wild-type level. The plasmid pKCA900, pSETA900, pLA900 and pLA400 joint transferred to the wild strain S.globisporusC-1027 to construct apoprotein corresponding over expression strain. Antibacterial activity and HPLC analysis showed that overexpression of cagA gene can increase the production of lidamycin.
The fusion protein expression plasmid pKCTH2600 to construct the apoprotein - single domain antibody, pSETTH2600 was injected into the apoprotein blocking lines for expression. The antibacterial activity of recombinant strain S.globisporus AKO/pKCTH2600 fermentation broth in the fermentation stage and wild strains, by Western blot analysis in intracellular, extracellular were detected specific fusion protein bands.ELISA detected the immunological activity of the fusion protein. The results showed that the fusion protein blocked in the apoprotein by multicopy plasmid form strains obtained expressed significantly more C-1027 gene engineering strain S.globisporus V_H and S.globisporus C-1027 NV_H.
In summary, the expression of the fusion gene by optimizing strategy, improve the expression level of fusion protein, to obtain anti type IV collagenase single domain antibody V_H oriented lidamycin laid the foundation for further construction of the high expression oriented lidamycin recombinant strains provide a new technology platform.

【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R392

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