抗IV型胶原酶单域抗体V_H导向的力达霉素基因工程菌株的构建及融合蛋白CagA-V_H的表达研究

发布时间：2018-03-15 08:05

本文选题：胶原酶　切入点：单域　出处：《中国协和医科大学》2009年博士论文　论文类型：学位论文

【摘要】： 力达霉素(lidamycin,LDM),原名C-1027,是一种新型的烯二炔类抗肿瘤抗生素,由球孢链霉菌C-1027(Streptomyces globisporus C-1027)产生。其抗肿瘤活性比临床常用的阿霉素强10,000倍,已经进入临床Ⅱ期试验。力达霉素由一个酸性辅基蛋白CagA(C-1027 apoprotein、C-1027AG,也写作LDP)和一个烯二炔结构的发色团(chromophore,也写作LDC)非共价结合组成,发色团为活性组分,辅基蛋白保护和运载发色团。由于力达霉素自身具有辅基蛋白,可以将其与抗肿瘤抗体偶联成为融合蛋白,改造为靶向特异肿瘤的载体,构建导向性力达霉素。Ⅳ型胶原酶降解细胞基底膜中的Ⅳ型胶原,破坏基底膜及细胞外基质的完整性,引起肿瘤细胞的侵袭与转移,Ⅳ型胶原酶已经成为抗肿瘤研究的靶标。抗Ⅳ型胶原酶抗体可以抑制Ⅳ型胶原酶的活性并可作为导向药物的载体。本工作根据已知的抗Ⅳ型胶原酶单链抗体基因序列,设计合成了具有链霉菌偏好密码子的抗Ⅳ型胶原酶单域抗体V_H(抗Ⅳ型胶原酶抗体重链可变区)基因,构建了含有辅基蛋白与抗Ⅳ型胶原酶单域抗体融合蛋白基因cagA-V_H和用于筛选的阿普霉素抗性基因aac(3)Ⅳ的重组质粒pBSH,用接合转移的方法导入力达霉素产生菌S.globisporus C-1027,通过同源重组双交换,以cagA-V_H -aac(3)Ⅳ取代力达霉素生物合成基因簇中原有的辅基蛋白基因cagA,得到基因工程菌株S.globisporus C-1027 V_H,以获得Ⅳ型胶原酶靶向的力达霉素。对基因工程菌株进行发酵,以枯草杆菌(Bacillus subtilis)为检定菌,结果表明基因工程菌株发酵液具有一定的抑菌活性,但与原产生菌相比活性较低。经Western blot分析在胞内能检测到融合蛋白和降解的辅基蛋白,而胞外仅检测到降解的辅基蛋白条带。ELISA检测到融合蛋白的免疫活性。同时构建了含有融合蛋白基因cagA-V_H但不含抗性基因的重组质粒pBS03H,通过同源重组双交换,以cagA-V_H基因取代cagA基因,得到仅融合了抗Ⅳ型胶原酶单域抗体基因的基因工程菌株S.globisporus C-1027NV_H。同样进行了发酵、抑菌活性、SDS-PAGE和Western blot的检测,结果与S.globisporus C-1027 V_H基本相同。为了增加融合蛋白的表达量,获得高活性的导向性力达霉素,本工作进一步构建了辅基蛋白阻断株,并通过导入多拷贝的融合蛋白表达质粒的策略来提高融合蛋白的表达量。构建重组质粒pBSA,将质粒接合转移至S.globisporus C-1027中,采用同源重组双交换的方法将cagA基因阻断。通过抗性筛选、PCR和Southern blot验证,最终获得辅基蛋白阻断株,命名为S.globisporus AKO。用四种辅基蛋白表达质粒pKCTA900、pSETTA900、pLTA900和pLTA400分别导入在自身启动子和/或强启动子控制下的cagA基因对阻断突变株S.globisporus AKO进行互补,得到相应的互补菌株。对阻断株和互补菌株进行发酵,发酵液的抑菌活性和HPLC分析结果表明阻断株完全丧失了力达霉素的产生能力,而互补菌株能不同程度地恢复产生力达霉素,其中S.globisporus AKO/pKCTA900基本恢复到野生株的水平。将质粒pKCA900、pSETA900、pLA900和pLA400接合转移至野生株S.globisporusC-1027中构建了相应的辅基蛋白过表达菌株。抑菌活性和HPLC分析结果表明过量表达cagA基因可以提高力达霉素的产量。构建辅基蛋白-单域抗体的融合蛋白表达质粒pKCTH2600、pSETTH2600分别导入辅基蛋白阻断株中进行表达。重组菌株S.globisporus AKO/pKCTH2600发酵液的抑菌活性在发酵晚期与野生株相当,经Western blot分析在胞内、胞外均能检测到融合蛋白的特异条带。ELISA检测到融合蛋白的免疫活性。结果表明融合蛋白在辅基蛋白阻断株中以多拷贝质粒的形式获得表达且表达量较基因工程菌株S.globisporus C-1027 V_H和S.globisporus C-1027 NV_H明显提高。综上所述,本工作通过优化融合基因的表达策略,提高了融合蛋白的表达水平,为获得抗Ⅳ型胶原酶单域抗体V_H导向的力达霉素奠定了基础,为进一步构建高表达的导向力达霉素重组菌株提供了新的技术平台。
[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.

【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

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