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

【摘要】： 力達(dá)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(chǎn)生。其抗腫瘤活性比臨床常用的阿霉素強(qiáng)10,000倍,已經(jīng)進(jìn)入臨床Ⅱ期試驗(yàn)。力達(dá)霉素由一個(gè)酸性輔基蛋白CagA(C-1027 apoprotein、C-1027AG,也寫作LDP)和一個(gè)烯二炔結(jié)構(gòu)的發(fā)色團(tuán)(chromophore,也寫作LDC)非共價(jià)結(jié)合組成,發(fā)色團(tuán)為活性組分,輔基蛋白保護(hù)和運(yùn)載發(fā)色團(tuán)。由于力達(dá)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(gòu)建導(dǎo)向性力達(dá)霉素。Ⅳ型膠原酶降解細(xì)胞基底膜中的Ⅳ型膠原,破壞基底膜及細(xì)胞外基質(zhì)的完整性,引起腫瘤細(xì)胞的侵襲與轉(zhuǎn)移,Ⅳ型膠原酶已經(jīng)成為抗腫瘤研究的靶標(biāo)�？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(dǎo)向藥物的載體。 本工作根據(jù)已知的抗Ⅳ型膠原酶單鏈抗體基因序列,設(shè)計(jì)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(gòu)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(zhì)粒pBSH,用接合轉(zhuǎn)移的方法導(dǎo)入力達(dá)霉素產(chǎn)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(dá)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(dá)霉素。對(duì)基因工程菌株進(jìn)行發(fā)酵,以枯草桿菌(Bacillus subtilis)為檢定菌,結(jié)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(chǎn)生菌相比活性較低。經(jīng)Western blot分析在胞內(nèi)能檢測(cè)到融合蛋白和降解的輔基蛋白,而胞外僅檢測(cè)到降解的輔基蛋白條帶。ELISA檢測(cè)到融合蛋白的免疫活性。同時(shí)構(gòu)建了含有融合蛋白基因cagA-V_H但不含抗性基因的重組質(zhì)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(jìn)行了發(fā)酵、抑菌活性、SDS-PAGE和Western blot的檢測(cè),結(jié)果與S.globisporus C-1027 V_H基本相同。 為了增加融合蛋白的表達(dá)量,獲得高活性的導(dǎo)向性力達(dá)霉素,本工作進(jìn)一步構(gòu)建了輔基蛋白阻斷株,并通過導(dǎo)入多拷貝的融合蛋白表達(dá)質(zhì)粒的策略來提高融合蛋白的表達(dá)量。構(gòu)建重組質(zhì)粒pBSA,將質(zhì)粒接合轉(zhuǎn)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(yàn)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(dá)質(zhì)粒pKCTA900、pSETTA900、pLTA900和pLTA400分別導(dǎo)入在自身啟動(dòng)子和/或強(qiáng)啟動(dòng)子控制下的cagA基因?qū)ψ钄嗤蛔冎闟.globisporus AKO進(jìn)行互補(bǔ),得到相應(yīng)的互補(bǔ)菌株。對(duì)阻斷株和互補(bǔ)菌株進(jìn)行發(fā)酵,發(fā)酵液的抑菌活性和HPLC分析結(jié)果表明阻斷株完全喪失了力達(dá)霉素的產(chǎn)生能力,而互補(bǔ)菌株能不同程度地恢復(fù)產(chǎn)生力達(dá)霉素,其中S.globisporus AKO/pKCTA900基本恢復(fù)到野生株的水平。將質(zhì)粒pKCA900、pSETA900、pLA900和pLA400接合轉(zhuǎn)移至野生株S.globisporusC-1027中構(gòu)建了相應(yīng)的輔基蛋白過表達(dá)菌株。抑菌活性和HPLC分析結(jié)果表明過量表達(dá)cagA基因可以提高力達(dá)霉素的產(chǎn)量。 構(gòu)建輔基蛋白-單域抗體的融合蛋白表達(dá)質(zhì)粒pKCTH2600、pSETTH2600分別導(dǎo)入輔基蛋白阻斷株中進(jìn)行表達(dá)。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(dāng),經(jīng)Western blot分析在胞內(nèi)、胞外均能檢測(cè)到融合蛋白的特異條帶。ELISA檢測(cè)到融合蛋白的免疫活性。結(jié)果表明融合蛋白在輔基蛋白阻斷株中以多拷貝質(zhì)粒的形式獲得表達(dá)且表達(dá)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(dá)策略,提高了融合蛋白的表達(dá)水平,為獲得抗Ⅳ型膠原酶單域抗體V_H導(dǎo)向的力達(dá)霉素奠定了基礎(chǔ),為進(jìn)一步構(gòu)建高表達(dá)的導(dǎo)向力達(dá)霉素重組菌株提供了新的技術(shù)平臺(tái)。
[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(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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