Construction, screening and immunogenicity of the recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2 / 细胞与分子免疫学杂志

Objective To construct a recombinant poxvirus vector vaccine, rVTTδTK-RBD, and to evaluate its safety and immunogenicity. Methods The receptor-binding domain (RBD) gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE, to construct the recombinant poxvirus shuttle vector pSTKE-RBD. This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain (VTT). The recombinant poxvirus rVTTδTK-RBD was successfully obtained after several rounds of fluorescence phage screening. The effect of rVTTδTK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination. The levels of specific and neutralizing antibodies produced by rVTTδTK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly. The effect of rVTTδTK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry. Results Through homologous recombination, enhanced green fluorescent protein (EGFP) screening marker, and multiple rounds of fluorescent phosphorescence phage screening, a recombinant poxvirus rVTTδTK-RBD, expressing RBD with deletions in the thymidine kinase (TK) gene, was successfully obtained, which was validated by PCR. The in vivo experiments on BALB/c mice showed that rVTTδTK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT. Conclusion The recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2 is successfully constructed and obtained, with its safety and immunogenicity confirmed through various experiments.

Isolation and Identification of Streptomyce from Cow Dung and Antimicrobial Activity of Its Substance / 医药导报

Objective To isolate and identificate Streptomyce strain from cow dung and observe its antimicrobial activity. Methods Strains were isolated from cow dung by dilution coating method.Strong antibacterial strains were screened out by agar block method with fixed Staphylococcus aureus and Escherichia coli as indicative bacteria.The strains were identified based on physiological and biochemical characteristics as well as 16S rDNA gene sequence analysis.Active antibacterial fermentation broth substance was determined by disk diffusion method,and antibacterial active substance of strains fermentation broth was extracted by water-saturated n-butanol.Antibacterial substance of strains was identified by Molish reaction,biuret test and ninhydrin reaction. Results Eight strains were isolated from cow dung and one strong antibacterial strain was screened out and named B5-2,identified as Streptomyces.The results showed that the strain had the highly antibacterial effect on Staphylococcus aureus,Escherichia coli,Citrobacter freundii,Enterobacter cloacae,Klebsiella pneumoniae,Enterobacter aerogenes.The strain antibacterial active substance of fermentation broth preliminary analysis showed that strong antibacterial active substance of B5-2 was the water-soluble substance.Antibacterial substance of B5-2 was preliminarily identified as glycoside and protein by Molish reaction,biuret test and ninhydrin reaction. Conclusion The strain isolated have a strong inhibition effect on clinical pathogenic bacteria in clinical practice.

Isolation,induced culture and identification of immature dendritic cells in vitro / 中国免疫学杂志

Objective:To establish a fast method for the generation of immature dendritic cells(DCs) from human peripheral blood mononuclear cells(hPBMCs) in vitro.Methods: High purity human CD14+ monocytes were collected using density Ficoll gradient centrifugation and MACS beads sorting system.iDCs(Immature DC) were induced after cultured with rhGM-CSF and rhIL-4 on the fourth day.Fluorescence activated cell sorting(FACS) was used to identify cell surface markers(CCR5) and capabilities of antigen uptake of iDCs on the fourth day.Ordinary optical microscope,scanning electron microscopy and transmission electron microscopy were used to observe surface and internal structure of iDCs on the fourth day of culture conditions.Results: FACS result shows that the purity of CD14+ monocytes collected from hPBMCs were more than 94%.The antigen uptake capability and CD195 of iDCs was detected on the fourth day of cultured conditions.Typical surface and internal structure characteristics of iDCs were observed.Conclusion: Rapid induction culture is an effective method for obtaining a large number of iDC with typical characteristics in vitro,and can be used for further experimental study.

Preparation and identification of Env-pseudotyped subtype B HIV-1 / 军事医学

Objective To prepare the Env-pseudotyped subtype B HIV-1 with enhanced green fluorescent protein ( EG-FP) gene,explore HIV-1 infection mechanisms and develop feasible methods of identification .Methods The Env-pseudo-typed viruses were packaged in HEK293T cells by cotransfection, and the reporter gene and P24 protein were detected by PCR, Western blot and ELISA .Reporter gene amplification , viral titration assay and a single round of infection assay were performed after the env-pseudotyped viruses infected HIV-1 permissive cell .Results and Conclusion A generation and identification method of the pseudotyped HIV-1 was established . The Env-pseudotyped subtype B HIV-1 has been prepared, which is able to infect SupT1 and TZM-bl cells through infection assay .

Inhibition effect on prostate cancer cells by an hTERT-promoter-dependent oncolytic adenovirus that expresses apoptin / 中华泌尿外科杂志

Objective To investigate the inhibition effects of an hTERT-promoter-dependent oncolytic adenovirus Ad-VT that expresses apoptin on human prostatic carcinoma cell PC-3. Methods MTT assay was used to measure viability of PC-3 cell which was infected by recombinant adenovirus.The viability was measured at time points of 12,24,36,48,60,72,84 and 96 h after infection.AO/EB staining,DAPI staining,Annexin V assay were used to investigate the lethal effect and style of Ad-VT on PC-3 cell in vitro.The Caspases were measured by whole cell extraction of PC-3 cells 48hrs after infection. Results Ad-VT,Ad-VP3 and Ad-GT inhibited the proliferation of PC-3 cell in vitro.Ad-VT and Ad-GT were more effective than Ad-VP3 on cell growth,P ＜ 0.05.At 48,72,96 h time points,the inhibition effect of Ad-VT on PC-3 cell exhibited a dose related manner.When infection at MOI 100,the inhibition effect of Ad-VT on PC-3 cells exhibited time related manner.The AO/EB staining,DAPI staining,Annexin V assay,Annexin V assays and Caspase assays showed that Ad-VT inhibited the proliferation of PC-3 cells by inducing apoptosis of prostate cancer cells,Loss of cytoplasmic membrane integrity. Conclusions The hTERT-promoterdependent oncolytic adenovirus Ad-VT could effectively suppress prostate cancer cells PC-3 growth.

Construction, expression and characterization of a dual cancer-specific fusion protein targeting carcinoembryonic antigen in intestinal carcinomas.

The abundantly expressed carcinoembryonic antigen (CEA) on several cancer types is an attractive target for antibody-directed therapy. However, CEA is also present in some normal tissues. Here, we produced a dual functioning protein, designated as CAtin that exhibits both specific binding and killing functions, by fusing a tumor-specific apoptosis-inducing molecular Apoptin to C-terminus of an anti-CEA single-chain disulfide-stabilized Fv antibody (scdsFv). The CAtin proteins were expressed in Escherichia coli (E. coli), refolded and purified on an immobilized Ni2+ affinity chromatography column. SDS-PAGE and Western blotting revealed that the recombinant protein was well-expressed and the yield was approximately 250mg/L. We demonstrated by flow cytometry and immunofluorescence assays that CAtin could bind specifically to human colon carcinoma cells (LoVo), but almost not to human uterine cervix (Hela). The results suggest that CAtin is active and specific toward CEA-positive cells and may potentially be used in CEA-targeted cancer therapy.

Prediction of the Th/B cell epitopes on HA of influenza virus(H1N1)and antigenicity analysis / 中国免疫学杂志

Objective:To predict Th/B cell epitopes in HA of influenza virus(H1N1)and analyze antigenicity of the candidate epitopes in order to develop epitope-bacterin by the way of bioinformatics.Methods:The HA amino acid sequences of infiuenza virus(H1N1),which the viral infection was prevalent recently,were downloaded from Genbank.The Th/B cell epitopes were predicted and analyzed by bioinformatics methods.Then,specificity and conservation of the candidate epitopes were estimated.Finally,antigenicity of the candidate epitopes was identified by influenza virus(H1N1)positiVe serum samples of mice.Results:Three Th/B cell epitopes containing HA_(73-87),HA_(125-139),HA_(188-205) were acquired Two of the candidate epitopes were in a relatively conserved domain of HA1,and a deal of 2006-2009 influenza virus(H1N1)isolates contained the sequences.Moreover,the candidate epitopes were showedin a distinct antibody combining reactivity with the influenza virus (H1N1)positive serum of mice,which inferred the predicted epitopes to be functional ones.Conclusion:The selected epitopes are able to be functional HA Th/B cell epitopes of influenza virus(H1N1).Our study also establish the foundations for the further research of influenza virus infectlon and immunity mechanism,the recognition of influenza virus(H1N1)functional epitope and the development of epitope vaccines.

Anti-tumor effects on human laryngeal carcinoma Hep-2 of recombinant fowlpox virus expressing chicken anemia virus Apoptin gene / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the anti-tumor effects and the mechanism of the recombinant fowlpox virus expressing Apoptin gene on human laryngeal carcinoma Hep-2.@*METHOD@#Hep-2 cells cultured in vitro were infected with vFVApoptin. The anti-tumor effects on Hep-2 cells were measured through MTT staining and, the mitochondrial trans-membrane potential (delta psi m) and reactive oxygen species (ROS) were analyzed by flow cytometry. Western blot was used to detect the release of cytochrome c (Cyto c). Caspase-3/9 activities were measured by colorimetric assay.@*RESULT@#vFVApoptin could restrain Hep-2 cells significantly and, had the function of down-regulating delta psi m, up-regulating ROS, promoting Cyto c release and activating Caspase-3/9.@*CONCLUSION@#Cyto c were released from mitochondria by the function of up-regulating ROS of vFVApoptin. Cyto c triggered Caspase-9 and, after the activation of Caspase-9, downstream apoptotic factors, such as caspase-3, were activated. Eventually, Hep-2 cells were suppressed by mitochondrial pathway apoptosis induced by vFVApoptin.

Molecular design and immunogenicity of a multiple-epitope foot-and-mouth disease virus antigen, adjuvants, and DNA vaccination / 生物工程学报

We designed and constructed a fuse expression gene OAAT and staphylococcal enterotoxin A (SEA) on the basis of the OAAT designed and constructed which consists of the structural protein VP1 genes from serotypes A and O FMDV, 5 major VP1 immunodominant epitopes from two genotypes of Asia1 serotype, and 3 Th2 epitopes originating from the non-structural protein, 3ABC gene and structural protein VP4 gene. The recombinant plasmids pEA was constructed using SEA as a genetic adjuvant. Expressions of target gene from the pEA in Hela cell were verified by IFA and Western blotting. The experiment of BALB/c mice immunized with the DNA vaccines showed that pA and pEA could induce simultaneously specific antibodies against serotypes A, Asia1, and O FMDV, and the highest antibody titres were found in the pEA and inactivated vaccine groups compared to pA vaccinating mice. Compared with the control, the levels of IL-2, IFN-gamma, IL-4, and IL-10 expression by splenic lymphocytes from mice immunized with pA and pEA were significantly increased. In addition, we found that the levels of IL-2, IFN-gamma and IL-4 from the mice immunized with pEA was higher than mice immunized with pA did. The results of viral challenge in guinea pigs showed the pA, pEA and inactivated vaccine provided full protection in 2/4, 2/4, 3/4, 3/4 and 4/4, 4/4 guinea pigs from challenge with FMDV O/NY00 and Asial/YNBS/58, respectively. The results demonstrated fuse protein OAAT and SEA may be potential immunoge against FMDV, furthermore, SEA may be an effective genetic adjuvant for DNA vaccine.

Preparation of recombinant human zona pellucida 3 protein and its immunologic activity / 中国免疫学杂志

Objective:To prepare and purify recombinant human zona pellucida 3 protein,and to study its immunologic activity.Methods:The E.coli BL21 containing recombinant plasmid pGEX4T-1/ huZP3 was induced to express GST-fusion protein by IPTG.After a series of purification procedure,the purified protein was analyzed by SDS-PAGE.After the mice immunized with rhuZP3,the antibody responses against rhuZP3 were detected by ELISA.Results:The soluble fusion protein was expressed,and purity of rhuZP3 was 95%.Moreover, purity of rhuZP3 could be recognized by anti-human ZP3 in ELISA.Conclusion:The rhuZP3 is obtained through the preparation of prokaryotic expression system and anti-rhuZP3 antibody has immunological activity.

Immunogenicity of plasmids encoding P12A and 3C of FMDV and swine IL-18.

In this paper, two recombinant plasmids (pVIR-P12AIL18-3C and pVIR-P12A-3C) containing foot and mouth disease virus (FMDV) capsid polypeptide, 3C coding regions of O/NY00 and using/or not swine IL18 as a genetic adjuvant were constructed, and evaluated for their ability to induce humoral and cellular responses in mice and swine. In addition, the ability to protect swine against homologous virus challenge was examined. Mice and swine were given booster vaccination twice and once, respectively, and swine were challenged 10 days after the booster vaccination. Control groups were inoculated with pVAX1 or phosphate-buffered saline (PBS). All animals vaccinated with pVIR-P12AIL18-3C and pVIR-P12A-3C developed specific anti-FMDV ELISA antibody and neutralizing antibody and T lymphocyte proliferation and CTL cytotoxic activity was observed. In addition, we found that pVIR-P12AIL18-3C possessed stronger immunogenicity than pVIR-P12A-3C. The pVIR-P12AIL18-3C and pVIR-P12A-3C provided full protection in 3/4 and 2/4 swine from challenge with FMDV O/NY00, respectively. The results demonstrate the potential viability of a DNA vaccine in the control and prevention of FMDV infections.

Construction and immunogenicity of recombinant fowlpox vaccines coexpressing HA of AIV H5N1 and chicken IL18.

cDNAs of the HA genes of subtype H5N1 AIV were fused to form a single open reading frame, designated H5HA-H7HA. The H5HA-H7HA cDNA and chicken Interleukin-18 (IL18) were inserted into the fowlpox virus (FPV) expression vector pUTA-16-LacZ to produce pUTAL-H5-H7-IL18. cDNA of H5N1 AIV HA was inserted into the FPV expression vector pUTA2 to create the recombinant expression plasmid pUTA2-H5. Plasmids were then co-transected into CEF cells. The two recombinant fowlpox viruses (rFPV) were produced by three cycles with the BrdU and verified by RT-PCR, IFA and Western blotting. One-day-old specific pathogen free (SPF) chickens and 7-day-old commercial Leghorn egg-laying chickens were inoculated with 10(6) PFU recombinant or parental fowlpox vaccine viruses by wing-web puncture. Hemagglutination inhibition (HI) antibody titer and nonspecific cellular immunity level were assessed after 1-3 weeks post-immunization. We found that all rFPV-vaccinated groups produced HI-specific antibodies, and the level of cellular immunity induced by the rFPV-H5-H7-IL18 strain was significantly higher than that induced by rFPV-H5HA. At 3 weeks post-inoculation, immunized SPF and Leghorn chickens were challenged with H5N1 HP AIV. The rFPV-H5-H7-IL18 vaccine strains were able to induce complete (10/10) protection, while the rFPV-H5HA vaccine strain induced (9/10) protection. Cloacal swabbing samples were collected from immunized leghorn chickens during the first week post-challenge; no shedding was found in the rFPV-H5-H7-IL18 vaccinated group. The rFPV-H5-H7-IL18 vaccinated group displayed significantly increased weight gain relative to the rFPV-H5HA group. This study reports a significant step in the further development of new AIV vaccines.

Immune responses of a designed HIV-1 DNA vaccine on rhesus monkeys.

An effective HIV-1 vaccine will be the ultimate solution for the prevention of HIV/AIDS, though HAART plays important roles in treating the disease. In this study, a large-scale recombinant DNA plasmid containing a designed HIV-1 multi-epitope-p24 chimeric gene was prepared and purified. Rhesus monkeys were then inoculated muscularly with the plasmid for four times in week 0, 4, 8 and 18. Whole blood was collected two weeks after the third and fourth inoculation, followed by serum and peripheral blood mononuclear cell (PBMC) separation. The CTL activity and proliferation of PBMCs stimulated by macaque MHC-I-restricted HIV-1 CTL epitope peptide were analyzed by MTT and LDH release assay, respectively. Th1 cytokines in supernatant of cultured PBMC stimulated by HIV-1 CTL epitope peptide and anti-HIV-1 antibody in serum were assayed by ELISA. The results showed that increased CTL target-killing activity, higher secretion of Th1 cytokines (IFN-γ and IL-2) and promoted proliferative reaction of monkey PBMCs stimulated by HIV-1 CTL epitope peptide were detected in the immunization group inoculated by the recombinant DNA vaccine for three times, which were further enhanced by the fourth inoculation. At the same time, HIV-1 specific antibody in serum of immunized monkeys was higher than that in controls. We concluded that the designed HIV-1 DNA vaccine may induce HIV-1 specific cellular and humoral immunity on monkeys.

Antileukemia immune response activated by interleukin-18 transfected dendritic cells of chronic myelogeous leukemia / 吉林大学学报(医学版)

Objective To study the antileukemia immune response of IL-18 gene transfected dendritic cells(DCs) of chronic myelogeous leukemia(CML).Methods DCs were transfected with IL-18 gene by liposomes in CML.The expression of IL-18 in IL-18 transfected DCs was detected.The percentages of CD80+ and CD86+ cells in IL-18 tranfected DCs were determined by FCM.The proliferation of T cell,NK and specific CTL kill activity induced by IL-18 gene transfected DCs were detected.Results The quantity of IL-18 in IL-18 tranfected DCs was(596?34.1)pg/2?106cells/48 h,while the culture medium of mock-transduced DCs and DCs did not secrete detectable levels of IL-18.The percentages of CD80+ and CD86+ cells in IL-18 tranfected DCs were higher than that in mock-transfected DCs(P

Overexpression of HIV-1 P17 protein in Escherichia coli and its purification / 吉林大学学报(医学版)

Objective:To highly express HIV-1p17 in E.coli and purify the protein.Methods:①Recombinant plasmid was constructed by inserting HIV-1p17 gene amplified by PCR into plasmid vector,pET28c;②The recombinant plasmid was expressed in BL21,BL21(DE3),BL21(DE3) plysS and HMS174(DE3) of E.coli separately;③The target protein were purified with Ni-NTA resin;④The purified protein was detected by western blot and ELISA.Results:The expression of the P17 protein in BL21(DE3) represented up to 32% of total protein in E.coli,which was the most amounts compared with other kinds of E.coli.The purity of the purified protein reached 95%.The purified protein was recognized by HIV-1P17McAb as well as by HIV-1 positive serum.Conclusion:The recombinant plasmid is highly expressed in BL21(DE3) of E.coli that can be proceeded to the immunocompetence and the bioactivity research.The method of Ni-NTA resin is simple with low protein losing and high purity.And the purified p17 can be employed in early detection of HIV-1 infection and prediction of the clinical progression.

Immune response in mice innoculated by NDA vaccine expressing chimeric gene gag-gp120 of HIV-1 / 中国免疫学杂志

Objective:To detect the immune response in mice inoculated by DNA vaccine expressing chimeric gene gag gp120 of HIV 1.Methods:After BALB/C mice were immunized by the eukaryotic expression plasmid pVAXGE,the numbers of T lymphocyte subgroups of spleen,the killing rate of specific CTL from the immunized mice and the titers of serum antibodies during the different time were observed.Results:The lymphocytes of the immunized group by recombinant plasmid pVAXGE proliferated,the specific CTL killing rate of spleen was higher significantly than those of the control group( P

Construction of recombinant fowlpox virus with HIV-1Gag-gp120 fusion protein / 中国免疫学杂志

Objective:To express Gag gp120 fusion protein in the recombinant fowlpox virus.Methods:The Gag gp120 gene of HIV 1 was inserted downstream of the combined promoter in pUTAL,an expressing plasmid was constructed.By transfecting the plasmid into CEF cells pre infected with FPV 282E 4 strain,a recombinant fowlpox virus vUTALG was obtained by selecting in the presence of BUdR and subsequent blue plaque screening.Results:Western blot showed that the recombinant virus expressed the Gag gp120 fusion protein in the infected CEF cells.The virus like particles formed by Gag protein were observed under electron microscope.Mice were immunized with the recombinant virus.The results showed the recombinant virus could induce HIV 1 specific antibody response.Conclusion:The recombinant fowlpox virus could express the Gag gp120 fusion protein.

Apoptin Inducing Apoptosis in the Multidrug-Resistant Model of Human Osteosarcoma Cell Line / 中国肿瘤生物治疗杂志

Objective: To investigate the curative effects of apoptin on the multidrug-resistant model, of human osteosarcoma cell line (R-OS-732). Methods: 363 bpVP 3 gene was cloned into the vector pIRES1 and the recombinant eukaryon expression vector pIRVP3 was transfected into R-OS-732 cells with liposome. The expression of apoptin gene at transcription level was proved by RT-PCR. The pathological changes of the cells was observed by light microscope and electronmicroscope. The transfected R-OS-732 cells were collected after 48 hours. The genomic DNA extracted from the cells was observed by agarose gel electrophoresis. The apoptosis rate of the cells was analysed by flow cytometry. Results: The expression of apoptin gene at transcription level had been proved by RT-PCR. The construction changes of the two groups were obviously different under light microscope: most of R-OS-732 cells in the transfected group existed in form of being away from the bottom of the culture dish after 24 hours, in form of apoptosis after 48 hours and in form of necrosis after 72 hours; but those cells in the controlled group grow luxuriantly. And under electronmicroscope there was much change of cell nucleus between the two groups. DNA ladder of the transfected cells was observed through agarose gel electrophoresis only one band was observed in the controlled group; but bands of fragmented DNA observed in ositive control group. Flow cytometry analysis showed that the apoptosis rate of the cells in the transfected group was relatively higher than that of the controlls( P

Comparison Researches on the Mode of Cell Death Induced by NDV-Strain Changchun and NDV-Strain Siping / 中国肿瘤生物治疗杂志

Objective: To investigate the mode of cell death caused by NDV strain Changchun and NDV strain Siping. Methods: Plaque formation, cell suppression test, gel electrophoresis, and TUNEL assay were used after the cells were infected by NDV. Results: The apparently pathological changes were observed in chicken embryo fibroblasts, BHK, Hela, Hep 2, HCT and OS 732 tumor cells, but not in Wish cells. The higher suppressed effect on tumor cells was found in the NDV strain Changchun than that in the NDV strain Siping. There was no dose effect relationship between NDV and tumor cell suppression, only optimum dose NDV could cause maximal tumor cells inhibitory effect. Conclusion: The mode of cell death might be different after infection of NDV. The NDV strain Changchun killed tumor cells mainly through apoptosis, while the NDV strain Siping killed tumor cells mainly through necrosis. \[

Strategy on Enhancement of bFGF Gene Expression and Purification of Its Products / 中国肿瘤生物治疗杂志

Objective: To adjust the distance between SD sequence and ATG in the same expressive plasmid pLX1 to enhance expression of heterologous bFGF gene in E. coli. Methods: Adjusting the different distance between SD sequence and ATG by Klenow and Mung Bean Nuclease. SDS PAGE and Western blot showed the expressed protein bFGF in E.coli. bFGF proteins were purified by HPHIC, HPGFC and HAC. Biological activity was examined by MTT. Results: Recombinant plasmids pLX2, pLX3 were obtained and the expressive levels were 8.03%, 9.9% respectively. Also the purified bFGF was obtained by HPHIC, HPGFC, HAC and its ED 50 was 2.29 ng/ml. Conclusion: Increasing the bFGF gene dosage by adjusting the distance between SD sequence and ATG could increase the expression level of a desired protein.