Specific anti-tumor effect induced by attenuated Salmonella typhimurium vaccine expressing extracellular region of vascular endothelial growth factor receptor 2

The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1 VEGFR2(n1-7) was transformed into competent attenuated Salmonella typhimurium SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1 VEGFR2(n1-7). To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy (TEM) was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an en- hanced accumulation of CD8 cytotoxic T lymphocytes, as well as an increase in CD4 cells in the tumors of animals treated with the oral gene vaccine compared to tumors from control group mice. Ultrastructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the recombinant bacteria; the exogenous gene can de delivered to the host by attenuated Salmonella typhimurium to produce anti-tumor effect with no obvious cytotoxity to the host. In this study, it is established that attenuated Salmonella typhimurium could be used as a vector for oral gene vaccine, and our study provided a theoretical basis for the body distribution and the metabolism of the recombinant bacteria. This strategy may provide a simple, safe and effective way for the prevention and treatment of tumors.     

Prophylaxis of tumor through oral administration of IL-12 GM-CSF gene carried by live attenuated salmonella

A live attenuated AraA^- autotrophic mutant ofSalmonella typhimurium (SL3261) was used as carrier for eukaryotic expression vectors EGFPN1, pCMVmIL-12, pCMVhIL-12, pCMVmGM-CSF and pCMVhGM-CSF and was administered orally to BALB/c and C57BL/6 mice. After 6 weeks, these mice were challenged with 4T1 and Lewis tumor cells respectively. GFP expression and gene integration could be detected in mice’s livers, spleens, intestines, kidneys and tumors. The serum level of cytokines increased significantly in treated mice, so did the ratio of CD ₈ ⁺ /CD ₄ ⁺ , which resulted in the tumor regression and prolongation of the survival time of those mice. These researches laid an experimental foundation for the tumor gene therapy using live attenuated salmonella.     

Specific immune responses induced by a multi-epitope antigen of hepatitis C virus in mice and rabbits

Five highly conserved and immunogenic epitopes of hepatitis C virus (HCV) have been chosen to form a multi-epitope antigen gene and fused with β-galactosidase gene to express a hybrid GZ-PCX antigen, which could be specifically recognized by human HCV sera. High level of anti-GZ-PCX IgG has been induced when mice or rabbits were immunized with GZ-PCX antigen emulsificated with complete Freund’s adjuvant or mixed with killed attenuatedSalmonella typhimurium SL3261. The specific anti-GZ-PCX IgG reached a high titer of 10^-6, which remained for several months. Specific cytotoxic T lymphocyte (CTL) effects, delayed type hypersensitivity reaction (DTH) and proliferation of peripheral lymphocytes have been induced by GZ-PCX antigen or synthetic peptides. High level of anti-GZ-PCX slgG has been detected in mice’s intestinal washing fluids, which indicates that the antigen induced mucosal immunity as well as systematic immunity. The studies show that the HCV multi-epitope antigen induces high level of specific immune responses without obvious toxicity, which might be able to provide protectivity to any HCV genotypes and isolates.     

A novel gene <Emphasis Type="Italic">R049</Emphasis> identified in uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> provides partial protection in mice from colonization

Uropathogenic Escherichia coli (UPEC) is the most common causative organism of human urinary tract infection (UTI). Several UPEC virulence factors have been identified, but more are yet to be found. We previously identified a novel 789-bp-long DNA fragment (named R049) in UPEC strain 132 using a suppressive subtractive hybridization technique. In the present study, we used genome walking to elongate the sequence of this fragment to obtain the whole gene sequence and examined the role of this gene product in generating protective immunity. Through bioinformatic analysis, we predicted that this gene is a 1311-bp open reading frame (ORF), which we designated ORF_R049 (GenBank accession No.: EF488001). We further constructed a prokaryotic expression system to express full recombinant R049 protein and isolated and purified the protein through IPTG induction and nickel affinity chromatography. Using mouse immunosera generated by the purified protein, we confirmed the natural expression and outer membrane localization of the protein in wild-type strain UPEC132 by Western blotting. To test the potential of this protein as a vaccine candidate, we immunized mice with the recombinant protein before challenging them with UPEC132 through the urinary tract. The results showed significantly reduced bacterial colonization in the urine and kidneys of the immunization group compared with the control group. However, the degree of renal pathological damage was not significantly improved in the immunized mice. Our study has identified a novel gene of UPEC which can generate protective immunity against UTI. This novel gene provides a promising new vaccine candidate.     

携带大肠杆菌融合基因K88ac-STII的减毒鼠伤寒沙门疫苗菌的构建与鉴定

为构建表达大肠杆菌菌毛蛋白K88ac和热稳定肠毒素STII的无抗性减毒鼠伤寒沙门氏菌疫苗株,采用缺失腺苷酸环化酶基因(Δcya)、环腺苷酸受体蛋白基因(Δcrp)以及天冬氨酸β-半醛脱氢酶基因(Δasd)的鼠伤寒沙门菌(X4550)作为宿主,将编码K88ac-STII的融合基因插入Asd 组成型表达载体pYA3334中,通过2次转化引入宿主菌,成功构建了表达K88ac-STII融合基因平衡致死的减毒鼠伤寒沙门重组菌株S.typhimuriumX4550(pYA3334K88ac-STII),为防治仔猪腹泻提供了口服活菌疫苗候选株。     

Protection against malaria by immunization with plasmid DNA encoding cholera toxin B subunit andPlasmodium falciparum epitopes

Cholera toxin B subunit is a good carrier protein and an effective adjuvant which can boost both cellular and humoral immunity. DNA fragments encoding B cell, Th cell and CTL epitopes of P. falciparum CS, MSA-1, MSA-2 and RESA antigens were cloned down-stream of cholera toxin B subunit gene in the same reading frame. Another modification using IL2 as adjuvant was also made. High titer of anti-malaria epitopes antibodies and strong cellular immunogenicity were elicited after Balb/c mice were immunized three times with 100 μg recombinant plasmid DNA dissolved in 100 μL PBS. 200 vaccinees were challenged with mouse Plasmodium yoelli to investigate if cross protection existed. The protective efficacy was about 50%. And it is found that the protective efficacy is correlated with CTL activity which was considered to be the primary effects of anti-sporozoite protective immunity. Better results might be expected when the DNA vaccine candidates were applied to primates.     

Construction and anti-tumor effects of recombinant fowlpox virus expressing Newcastle disease virus hemagglutinin-neuramidinase gene

Hemagglutinin-neuramidinase (HN), a Newcastle disease virus-derived protein, not only mediates receptor recognition but also possesses neuraminidase (NA) activity, the ability to cleave a component of those receptors, N-acetylneuraminic acid (NAcneu, sialic acid). It is known that this protein in mammalian species, including human beings, has interesting anti-neoplastic as well as immune stimulating properties. To explore the use of the HN gene in cancer gene therapy, we constructed a recombi-nant fowlpox virus expressing the HN protein (vFV-HN) and compared the anti-tumor activity of the recombinant virus with that of wild-type fowlpox virus (FPV) in vivo and in vitro. Here we found that although B16 cells were somewhat resistant to the basal cytotoxic effect of wild-type fowlpox virus, infection with vFV-HN caused a pronounced cytotoxic effect and, the survival of tumor-bearing mice immunized with vFV-HN was significantly increased compared with the survival of mice immunized with the FPV alone. Furthermore, the immunization of mice with vFV-HN elicited a B16 tumor-specific cytotoxic T lymphocyte (CTL) response and clonal expansion of both CD4 and CD8 T cell populations in vivo. In addition, T cells from lymph nodes of mice vaccinated with vFV-HN secreted high levels of the Th1 cytokine IL-2 and IFN-γ, indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with vFV-HN may be a potential strategy for cancer gene therapy.     

HPV16E7-HSP70 Hybrid DNA Vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

0 IntroductionInbroicguhltat einxpge cDtaNtiAonvafcocri nper eivse ant ninegw aanpdp cruoraicnhg wdiits-heases . Due to the comparative weakness of cellulari mmunity that is purely induced by E7 DNA vac-cine,research on DNA vaccine is now focused onhowto enhance the i mmunogenicity of DNA vac-cine.It has been found in some research that thestrength of cellular i mmunity activity has things todo with the speed of antigen molecules’degrada-tion,i .e.the faster the degradation,the higher th…     

小鼠结核分枝杆菌感染模型用于评价重组HBHA疫苗免疫效果的实验研究

目的通过复制小鼠结核病模型评价重组HBHA疫苗的免疫效果。方法将重组肝素结合血凝素(HBHA)疫苗接种BALB/c小鼠,观察其诱导的细胞免疫应答水平,并以MTB H37Rv毒株攻击免疫小鼠,研究重组疫苗诱导的保护力。结果重组HBHA免疫BALB/c小鼠后可诱发细胞毒作用,将体内MTB裂解。重组疫苗不仅能刺激CD4+T,CD8+T增殖、活化,还可诱导脾细胞分泌IFN-γ、IL-2等细胞因子。H37Rv毒株攻击后,被免疫小鼠的组织病理学症状减轻。结论本研究通过检测小鼠体内细胞免疫应答和肺脏组织病理学改变等指标,客观的反映了重组疫苗免疫小鼠结核模型的保护效果。     

Expression ofBcl-2 in cells with different telomerase activities

Both telomerase andBcl-2 are important genes in controlling apoptosis. The activation of telomerase and the abnormal regulation ofBcl-2 are also closely related to carcinogenesis. However, little is known about the linkage between telomerase andBcl-2. The effect of activated telomerase on the expression ofBcl-2 has been investigated. It is demonstrated that in tumor and transformed cells with higher telomerase activity,Bcl-2 expression is significantly lower than that in telomerase negative or less telomerose activity cells. Further study showed that in the telomerase gene-transformed 2BS-fibroblasts,Bcl-2 expression is inhibited significantly while the exogenous telomerase catalytic subunit gene is re-expressed in fibroblasts. Results indicated that there might be a certain linkage between the expression of telomerase andBcl-2, and overexpression of exogenous telomerase gene might down regulate the expression ofBcl-2. They contributed equally to this work.     

Effects of soy isoflavone extracts on the growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in ovariectomized nude mice

In this study, we explored the effects of soy isoflavone extracts on the growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in ovariectomized athymic mice. The ovariectomized athymic mice were implanted with MCF-7 cells. They were fed with low, moderate and high doses of soy isoflavone extracts, at dietary concentrations of 6.25, 12.5 and 25 g/kg, respectively. The expression of ki-67 was detected by immunohistochemistry. The pS ₂ expression in tumors was analyzed by real-time PCR. Estrogen level in the serum was measured by chemiluminescence enzyme immunoassay. Compared with the control group, dietary soy isoflavone extracts had a significant stimulatory effect on MCF-7 tumor growth in mice (P < 0.05). The ki-67 and pS ₂ mRNA expressions in tumors were significantly increased by 6.25 and 12.5 g/kg dose of soy isoflavone extracts (P < 0.05). And, estrogen level in serum of 6.25 and 12.5 g/kg dose groups was higher than that of control group (P > 0.05). In conclusion, in the tested dietary concentration range soy isoflavone extracts had a stimulatory effect on tumor growth. 6.25 and 12.5 g/kg doses of soy isoflavone extracts can increase the cell proliferation in tumors and induce estrogen-responsive pS2 expression. Supported by National Natural Science Foundation of China (Grant No. 30572133)     

HIV-1C亚型DNA疫苗诱导小鼠免疫反应的研究

 构建含中国流行株HIV-1 C亚型核心蛋白gag基因的重组质粒pVAX-gag,并在体外进行了表达与鉴定.同时构建了含此gag基因的原核表达质粒pGEX-gag,表达纯化并鉴定重组蛋白Gag.以质粒pVAX-gag免疫Balb/C小鼠后,用ELISpot和流式细胞仪检测其细胞免疫反应.再以纯化后的重组蛋白Gag作为包被抗原,用ELISA检测其体液免疫反应.结果显示重组质粒pVAX-gag免疫小鼠后可有效地诱导机体产生细胞免疫和体液免疫反应,且免疫剂量和免疫效果存在一定的正相关性.重组原核表达质粒pGEX-gag的表达产物能与抗p24单克隆抗体发生特异性反应,可用于抗HIV抗体检测.     

Combined immunity of DNA vector and recombinant vaccinia virus expressing Gag proteins of equine infectious anemia virus

In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost procedures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag proteins. The results showed that the specific lysis of CTL responses in the DNA rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses induced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to produce powerful cellular responses. These results lay an important foundation for the development of a new EIAV genetic engineering vaccine.     

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the microparticle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 μg/mg soluble proteins on average and the highest value was 2.497 μg/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bioreactor producing high value materials such as edible HBV vaccine was discussed as well.     

P15——a new tumor suppressor gene

In addition to the tumor suppressor genes such as Rb and p53, it has been found that some molecules of the same class named CKI (cyclin-dependent kinase inhibitor) also play an important role in the inhibition of tumorigenesis and the tumor progression. In the KIP and INK4 families of CKIs, p15 shares extensive homology with p16. Findings in many tumors and their cell lines show that the inactivation of p15 (deletion, mutation, rearrangement, etc.) is very frequent, and inactive p15 is involved in the progress of some tumors. These studies provide evidence that the p15 is a new tumor suppressor gene. Furthermore, the research on the molecular mechanism of p15 in regulation of cell proliferation shows that p15 can inhibit the growth of some kinds of tumor cells, and p15 is the mediator of TGF-β-induced cell arrest. Investigations on p15 in cell differentiation suggest that increased p15 is related to the change of malignant phenotype. These results supply clues for further interpretation about the molecular mechnism of cell cycle control and cell tumorigenesis. And they may provide theoretical and experimental basis for application of p15 to clinical therapy of tumors.     

重组戊型肝炎疫苗的免疫原性试验

目的　研究重组戊型肝炎疫苗的免疫原性。方法　通过对小鼠、新西兰兔的多点皮内注射及肌肉注射 ,然后测定其抗体效价 ,研究重组戊型肝炎疫苗的性能。结果　小鼠在免疫 7d后开始产生抗体 ,两周抗体阳性率为80 % ,四周抗体阳性率达 10 0 %。新西兰兔从第二周后开始产生抗体 ,第三周达到高峰 ,抗血清在 1∶6 4 0 0稀释度的阳性率达 4 0 %。结论　重组戊型肝炎疫苗免疫动物具有良好的免疫原性 ,为进一步研究开发戊肝疫苗对人体的免疫提供了基础     

Construction and function of recombinant AcMNPV with double copies of v-cath gene

Two recombinant baculoviruses, dciAcMNPV and dcdAcMNPV in which another copy of the v-cath gene controlled by ie1 promoter and polh promoter was inserted, were respectively constructed by the Bac-to-Bac system. The expression of the v-cath gene of the recombinant baculoviruses in Sf9 cells at different phases was investigated by SDSPAGE and Western blot. The results showed that only recombinant virus dciAcMNPV containing late gene v-cath driven by early gene promoter could express V-CATH protein, cathepsin encoded by virus genome, 12 h post-infection and dcdAcMNPV containing late gene v-cath driven by late and very late gene promoters could express more V-CATH protein. Negative control ncAcMNPV, a mutant deleted vcath gene, could not express V-CATH protein at all. The Spodopera exigua larvae were infected with viruses respectively and the results showed that the toxicity was as follows: dcdAcMNPV>dciAcMNPV>wtAcMNPV>ncAcMNPV. The toxicity of recombinant viruses and the characters of dead larvae showed that the v -cath gene was relative to viral toxicity and host liquefaction. Recombinant baculovirus dcdAcMNPV might be used as a new kind of safe viral-pesticide, because of its high toxicity obtained by adding another gene copy and changing the expression level of its own gene relative to virulence.     

Deregulation of c-myc and SV40Tag causing brain tumor in mice

Deregulated expressions of both c-myc and simian virus 40 large T antigen （SV40Tag） are consistent features of lots of tumors. To investigate whether the expression of c-myc and SV40Tag in mouse might help develop a model of human tumor, we generated c-myctransgenics by inserting human c-mycgene into pTRE2 of Tet-On system. We obtained conditional expression of SV4OTag transgenics by the Tet-On system from Yangzhou University. Crossing the c-myc transgenic mouse with the SV40Tag transgenic mice to generate bitransgenics we got double-transgenic mice expressing c-myc and SV40Tag by the Tet-On system. After being treated with doxycycline continuously, single-transgenic SV40Tag mice developed brain tumor infrequently （3 of 84, 3.6%） with a long onset （185 d on average）. In contrast, double-transgenic c-mydSV4OTag mice developed brain tumor with a short onset （96 days on average） and a 41% brain tumor incidence rate （7 of 17, 41%）. This tumor was assumed to be medulloblastoma. Our experiments suggest that deregulated expression of c-myc and SV4OTag in brain might generate a mouse model of human brain tumor that recapitulates some features of human medulloblastoma.     

产丁醇大肠杆菌工程菌的构建

【目的】为了在大肠杆菌(Escherichia coli)中导入改良的丁醇合成途径,使非生产菌株大肠杆菌具备产丁醇的能力。【方法】克隆大肠杆菌乙酰转移酶基因atoB和丙酮丁醇梭菌(Clostridium acetobutylicum)丁醇合成途径关键酶基因(crt、hbd、adhE),构建多顺反子表达质粒pSE380-atoB-adhE-crt-hbd;克隆齿垢密螺旋体(Treponema denticola)反式烯酰辅酶A还原酶基因ter,构建表达质粒pSTV29-ter,并将双质粒导入到大肠杆菌。【结果】构建的工程菌能半厌氧发酵产微量丁醇,产量为0.08g/L。【结论】大肠杆菌中的丁醇合成途径导入成功,构建了产丁醇的大肠杆菌工程菌。     

Construction and<Emphasis Type="Italic">in vitro</Emphasis> study of an E1B-defective adenovirus

An E1B-defective adenovirus named rl/Ad was constructed by homologous recombination.The construction,selection and propagation of recombinant virus was done in the human embryonic kidney 293 cells (HEK293).The in vitro study demonstrated that the recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as the human glioblastoma tumor cells (U251) and human bladder tumer cells (EJ) but not in the normal cells with functional p53 such as the human fibroblast cells (MRC-5).Also,based on the cytopathic effect (CPE),it was demonstrated that the U251 cells were more sensitive to the infection of rl/Ad than that of EJ cells under identical conditions.In this paper,it was found that rl/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus.This may help to determine the safety of using any E1B-defective adenoviruses in cancer gene therapy.