| Lidamycin(C-1027),produced by Streptomyces globisporus C-1027,is a novel enediyne antitumor antibiotic and has entered phaseâ…¡clinical trial in China recently.Lidamycin comprise a 1:1 complex of an acidic apoprotein(CagA) and a noncovalently bound chromophore.Its anti-tumor activity is 10,000 times more potent than adriamycin used in clinical.The chromophore is the active component but highly labile and instable,and CagA protects and transports the chromophore.CagA could be coupled with the anti-tumor antibodies as a fusion protein to construct tumor-specific targeting antibiotic. Typeâ…£collagenase,degrading collagen typeâ…£in the cell basement membrane,plays an important role in tumor cell invasion and metastasis and is an attractive target for mAb-directed therapy.Anti-typeâ…£collagenase antibodies were employed in inhibition of the activity of typeâ…£collagenase and may act as a carrier of drug in cancer therapy.Based on the sequence of anti-typeâ…£collagenase single-chain antibody gene,its single domain antibody V_H(heavy chain variable fragment) gene containing the Streptomyces prefered codon was designed and synthesized.The recombinant plasmid pBSH containing the fusion protein CagA-V_H gene and apramycin resistance gene aac(3)â…£was constructed.Through conjugative transfer from E.coli ET12567/pUZ8002 into lidamycin producing strain S.globisporus C-1027,cagA-V_H-aac(3)â…£replaced cagA by homologous double crossover,resulting recombinant strain S.globisporus C-1027 V_H. The antibacterial activity of the recombinant strain was lower than the wild type strain in fermentation broth.Western blot analysis showed that fusion protein could be detected intracellularly but not extracellularly using CagA antibody.ELISA assay detected minimal immunological activity of the fusion protein CagA-V_H.Simultaneously,cagA gene was replaced by the cagA-V_H without aac(3)â…£,resulting S.globisporus C-1027 NV_H.Similar results of the fermentation,antimicrobial activity and Western blot analysis were obtained as S.globisporus C-1027 V_H.In order to increase the expression level of fusion protein,cagA gene disrupted strain AKO was constructed to be used as host for the fusion protein expression.Using homologous double crossover method to disrupt the cagA gene,S.globisporus AKO was obtained after PCR and Southern blot verification.Complementation of the S. globisporus AKO with four plasmids pKCTA900,pSETTA900,pLTA900 and pLTA400 was conducted respectively.The AKO and complementary stains were fermented,and the antibacterial activity and HPLC analysis showed that AKO completely lost the ability to produce lidamycin while complementary strains restored the lidamycin production in varying degrees.Within them,S.globisporus AKO/pKCTA900 produced lidamycin at wild type strain level.The plasmid pKCA900,pSETA900,pLA900 and pLA400 were conjugatively transferred to wild type strain of S.globisporus C-1027 to get overexpression strains.Antibacterial activity and HPLC analysis showed that overexpression of cagA gene can increase the production of lidamycin.Fusion protein CagA-V_H expression plasmid pKCTH2600 and pSETTH2600 were constructed and transferred into S.globisporus AKO.The antibacterial activity of recombinant strains of S.globisporus AKO/pKCTH2600 was similar as wild type strain in late fermentation stage.Western blot analysis showed that the fusion protein can be detected intracellularly and extracellularly,but the degradation of CagA was still detected.ELISA assay detected the immunological activity of the fusion protein.The results showed that the fusion protein expressed better in AKO/pKCTH2600 than the recombinant strains S.globisporus C-1027 V_H and S.globisporus C-1027 NV_H.In conclusion,this work improved the expression level of C-1027 apoprotein-V_H fusion protein by optimizing the expression strategy,and provided a convenient platform for the improvement of production of various tumor-targeting lidamycin.
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