Generation and evaluation of a recombinant myxomavirus expressing the VP60 protein of rabbit haemorrhagic disease virus / 生物工程学报

Rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV), are two pathogens that have harmful effect on rabbit breeding and population decline of European rabbits in their native range, causing rabbit haemorrhagic disease (rabbit fever) and myxomatosis, respectively. The capsid protein VP60 of the RHDV represents the major antigenic protein. To develop a recombinant bivalent vaccine candidate that can simultaneously prevent these two diseases, we used the nonessential gene TK (thymidine kinase) of MYXV as the insertion site to construct a recombinant shuttle vector p7.5-VP60-GFP expressing the RHDV major capsid protein (VP60) and the selectable marker GFP. Then the shuttle vector p7.5-VP60-GFP was transfected into rabbit kidney cell line RK13 which was previously infected with MYXV. After homologous recombination, the recombinant virus expressing GFP was screened under a fluorescence microscope and named as rMV-VP60-GFP. Finally, the specific gene-knock in and expression verification of the vp60 and gfp genes of the recombinant virus was confirmed by PCR and Western blotting. The results showed that these two genes were readily knocked into the MYXV genome and also successfully expressed, indicating that the recombinant MYXV expressing the vp60 of RHDV was generated. Protection against MYXV challenge showed that the recombinant virus induced detectable antibodies against MYXV which would shed light on development of the effective vaccine.

Adenovirus-mediated TK gene combined with α-IFN in treatment of nude mice transplanted with human renal clear-cell carcinoma / 第二军医大学学报

Objective: To investigate the inhibitory effect of adenovirus-mediated herpes simplex virus-thymidine kinase (TK) gene combined with α-IFN on renal clear-cell carcinoma. Methods: Adenovirus containing suicide gene TK, in combination with GCV or α-IFN, was used to treat human renal clear-cell carcinoma cell line 786-0, and the in vitro cytotoxic effects against 786-0 were evaluated using MTT method. The subcutaneous transplantation model of 786-0 cells was established with nude mice. Adenovirus containing TK gene was injected intratumorally and the GCV (50 mg/kg) was injected intraperitoneally; α-IFN (104 U/L) was injected intratumorally in combined therapy. The growth of tumors was observed after treatments. Results: The survival rate of 786-0 cells was (35.07 ± 1.43)% in the TK+GCV+α-IFN group, (68.57 ± 1.41)% in the TK+ GCV group and (68.65 ± 1.45)% in the α-IFN group (P=0.000). There was an obvious synergic effect between Ad-TK and α-IFN in inhibiting 786-0 cells. Ad-TK combined with GCV and α-IFN significantly suppressed the growth of 786-0 cells growth in nude mice model. Conclusion: Adenovirus-mediated TK plus prodrug GCV combined with α-IFN has obvious therapeutic effect in treatment of human renal clear-cell carcinoma.

Altered Expression of Connexin-43 and Impaired Capacity of Gap Junctional Intercellular Communication in Prostate Cancer Cells / 华中科技大学学报（医学）（英德文版）

Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elucidate the reason why the so-called "bystander effect" mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis.mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by reverse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malignant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. Assessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was abnormally located and markedly diminished as compared with normal prostatic epithelial ones, displaying a negative correlation to the pathological grade (χ2=4.025, P＜0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indicated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein participated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of "bystander effect", but also to initiation and progression of prostatic neoplasm.

Cooperative Therapeutic Effects of Herpes Simplex Virus Thymidine Kinase Gene/Ganciclovir System and Chemotherapeutic Agents on Prostate Cancer in vitro / 华中科技大学学报（医学）（英德文版）

The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer (HRPC) cells PC-3m were investigated. After transferring of the HSV-tk gene into PC-3m cells, mRNA and protein expression of HSV-tk was detected by reverse-transcript polymerase chain reaction (RT-PCR) and strept avidin-biotin complex (SABC) immunohistochemical method. The killing effect of GCV, cisplatin (CDDP), etoposide (VP-16), vincristine (VCR), methotrexate (MTX), 5-fluorouracil (5-Fu), and suramin on PC-3m cells was evaluated by morphological assessment analysis, trypan blue exclusion assay and MTT assay respectively. Additionally, the cooperative effect of HSV-tk/GCV system combined with the above agents on the target cancer cells was determined by MTT. Furthermore, apoptosis and necrosis induced by GCV plus 5-Fu or suramin was analyzed by flow cytometry (FCM). The results showed that that there was HSV-tk mRNA and protein expression in pDR2-tk plasmid transduced PC-3m cell. Combination of GCV with VP-16, VCR, 5-Fu or suramin led to an enhanced cellular killing effect, but with CDDP resulted in a reduced one and with MTX in an approximate one. FCM revealed that synergistic use of GCV and 5-fu or suramin resulted in a rather large proportion of apoptosis and necrosis with the apoptosis index being 36.38 % and 35.51%, and the proportion of necrosis being 33.05 % and 28.87 %, respectively. In conclusion, HSV-tk/CGV approach by addition of certain clinical available chemotherapeutic drugs brings on statistically significant enhanced cell killing over single-agent treatment.Our results highlight the potential for such new combination therapies for future treatments of HRPC.

Detection of Glycoproteins (B and D) and Ttymidine Kinase Genes of Herpes simplex virus Type 2 Strain G

BamHI restriction patters and genomic library of Herpes simplex virus type 2 (HSV-2) stram G were constructed, and locations of the glycoproteins gB and gD, and it genes on the fragments were detected by Southern blot analysis. HISV-2 genomic DNAs were cleaved into twenty-seven fragments by BamHI enzyme in the range of 0.72 to 15.08 (total 150.44 kb), which were cloned into the BamHI site of pBluescript SK(+) to construct genome library of the HSV-2. The library was named by the order of the fragment size from smallest one to largest one. HSV-2 glycoprotein gD gene was located in PHLA2-21 and PHLA2-22 recombinant plasmids, gB gene in PHLA2-24 plasmic, and it gene in PHLA2-11 clone by Southern blot analysis.

Detection of Glycoproteins (B and D) and Ttymidine Kinase Genes of Herpes simplex virus Type 2 Strain G

BamHI restriction patters and genomic library of Herpes simplex virus type 2 (HSV-2) stram G were constructed, and locations of the glycoproteins gB and gD, and it genes on the fragments were detected by Southern blot analysis. HISV-2 genomic DNAs were cleaved into twenty-seven fragments by BamHI enzyme in the range of 0.72 to 15.08 (total 150.44 kb), which were cloned into the BamHI site of pBluescript SK(+) to construct genome library of the HSV-2. The library was named by the order of the fragment size from smallest one to largest one. HSV-2 glycoprotein gD gene was located in PHLA2-21 and PHLA2-22 recombinant plasmids, gB gene in PHLA2-24 plasmic, and it gene in PHLA2-11 clone by Southern blot analysis.

Cloning, Sequencing and Expression in Escherichia coli of Herpes simple virus Type-1 Thymidine Kinase Gene

Cloning, sequencing and expressing in E. coli of the thymidine kinase (TK) gene of Herpes simplex virus type-1 (HSV-1) strain F was investigated. The TK gene, located in the BamHI 3.74 kb DNA flagment of the plasmid PHLA-12, was amplified by polymerase chain reaction (PCR). The 1,131 kb PCR product was cloned into the BamHI and BglII sites of pQE-30, and named pQE-TK recombinant. The TK gene was subcloned into the BamHI and BglII sites of pQE-30, and named pQE-TK recombinant. The nucleotide sequence of the 1,131 kb TK gene was determined, and the GC content was 65.13%. There were deduced 367 amino acid residues with a total molecular weight of 43 kDa. The weight was confirmed by the protein produced by E. coli M15/pQE-TK on the SDS-PAGE and Western blot. The production of the TK protein in the IPTG induced cells was measured over 4 h. At the end of 1, 2 and 3 h the level increased by 146,204 and 242%, respectively. The amount of the protein at the highest fraction Purified with Ni-NTA resin chromatography was 0.68 ug Per ml. The soluble state TK protein was present in the cytoplasm. In these results the F strain was different in base sequence and amino acid sequence from that of the CL101 strain, which caused difference in their strains.

Adenovirus-mediated TK gene combined with ?-IFN in treatment of nude mice transplanted with human renal clear-cell carcinoma / 第二军医大学学报

Objective:To investigate the inhibitory effect of adenovirus-mediated herpes simplex virus-thymidine kinase (TK) gene combined with ?-IFN on renal clear-cell carcinoma. Methods: Adenovirus containing suicide gene TK, in combination with GCV or ?-IFN, was used to treat human renal clear-cell carcinoma cell line 786-0, and the in vitro cytotoxic effects against 786-0 were evaluated using MTT method. The subcutaneous transplantation model of 786-0 cells was established with nude mice. Adenovirus containing TK gene was injected intratumorally and the GCV (50 mg/kg) was injected intraperitoneally; ?-IFN (104 U/L) was injected intratumorally in combined therapy. The growth of tumors was observed after treatments. Results: The survival rate of 786-0 cells was (35.07?1.43)% in the TK+GCV+?-IFN group, (68.57?1.41)% in the TK+GCV group and (68.65?1.45)% in the ?-IFN group ( P=0.000). There was an obvious synergic effect between Ad-TK and ?-IFN in inhibiting 786-0 cells. Ad-TK combined with GCV and ?-IFN significantly suppressed the growth of 786-0 cells growth in nude mice model.Conclusion: Adenovirus-mediated TK plus prodrug GCV combined with ?-IFN has obvious therapeutic effect in treatment of human renal clear-cell carcinoma.