| The resurgence of interest in virotherapy over the course of the last decade has led to several promosing modalities for the treatment of human cancers. Among these are transcriptional regulated adenovirus variants that preferentially replicate in and target tumor cells. Additionally synergistic effects have been seen when oncolytic viruses have been used in combination with traditional chemotherapy or radiotherapy.By 2002, a variety of viruses, including over 50 tumor-selective oncolytic adenovirus variants, had been tested for antitumor activity in animals or human being. One of the approaches to construct these tumor-specific oncolytic adenovirus is to limit the expression of essential viral genes such as El only in tumor tissues through the use of tumor- or tumor-specific promoters. For example, Yu et al utilized prostate-specific transcriptional regulatory elements(TREs) to limit the E1a expression only to target prostate cancer, but keep the gene silent in normal tissue cells. (Ela functions to stimulate S-phase entry and transactivate both viral and cellular genes that are crucial for a productive viral infection). Except for PSA (prostate-specific Antigen), some other tumor-specific TRE has been identified, such as a-fetoprotein (AFP) for hepatocelluar carcinoma cell (HCC), human telomerase promoter, mouse tyrosinase promoter, carcinoembryonic antigen (CEA) promoter and enhancer et al.Here we construct a hTERT and Cox-2 promoter regulated oncolytic virus byreplace the endogenous adenovirus El a and Elb promoter respectively. Materials and Methods:For insertion of human TERT and Cox-2 promoter before the El a and Elb transcription start site, a unique Age I site was created in the adenovirus El a promoter of plasmid pXCI and a Eag I site in the Elb promoter site to construct a new plasmid named pd306(kindly presented by Dr. DC Yu). The plasmid of pd306 was digest by Age I and Eag I respectively and then de-phosphate by ALK phosphatase. Human genomic DNA extracted from a normal peripheral blood using QIAamp DNA Blood Mini Kit was used as template. According to Genbank, different primers of hTERT and Cox-2 promoter were designed for PCR reaction. The PCR products containing the hTERT and Cox-2 promoter was digested with Age I and Eag I respectively. And then the insert fragment was subcloned into pd306 by T4 DNA ligase. The clones was transformed into DH-5a. The cloned plasmids were identified by PCR, Age I and Eag I digest and sequence analysis.Result and discusstionWe got two different insert fragments of the hTERT and Cox-2's promoters by PCR reaction with different primers. The length of hTERT promoter is 181bp (from 181/-1), 378bp (from -378/-1) respectively. The hTERT promoter is GC rich and lacks both TATA and CAAT boxes. The 181bp of hTERT promoter is considered as the core promoter region upstream of the transcription start site, containing two major factors binding sites, an E box(CACGTG) binding factor and spl(CGGCCG).The two different length of Cox-2 promoter fragment is 409bp (-409/-1) and 814bp (-814/-1) respectively. The Cox-2 promoter have three important transcription regulation sites-nuclear factor k B (NF- k. B, -223/214 ). nuclear factor interleukin 6 (NF-IL6, -132/-124) and a cAMP responsive element overlapping a non-canonical E-box (CRE/E-box, -59A49). The promoter has typical TATA box.. Sequence analysis show the PCR products don't have mutation in important transcription regulation site.We subcloned the promoter sequences of hTERT into the plasmid pd306, which has been digested by Age I enzyme thoroughly, de-phosphated and be purified. The recombinant plasmids were transformed into the bacteria DH-5 a . The recombinant plasmids were identified by specific PCR reaction, Age I digestion and sequence analysis. Then the right clones were digested by Eag I enzyme, de-phosphated and purified by PCR purification kit. And different Cox-2 promote was inserted. Finally, the bacteria transformation and identification were performed same as before.Conclusion:1.
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