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Aim To investigate the effeets of Dracocephalum Moldavica total flavonoids (TFDM) on the foaming lipoprotein (ox-LDL), and to further elucidate the mechanism of anti-atherosclerosis (AS) of TFDM. Methods RAW264. 7 maerophages were cultured in vitro and induced to become foam cells by ox-LDL stimulation, and inflammation of mouse monocyte macrophage leukemia cells (RAW264. 7) induced by oxi-dized low density TFDM(25, 50, 100 mg • L"
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Objective:To investigate the influence of peer education based on WeChat support on the social support, mental flexibility and rehabilitation self-efficacy of patients after laparoscopic radical prostatectomy.Methods:A total of 82 patients after laparoscopic radical prostatectomy who were admitted from January 2018 to December 2019 were selected as the research objects. According to the operation time, they were divided into the treatment group (January 2019-December 2019) with 43 cases and the control group (January 2018-December 2018) with 39 cases. The control group was given regular health education, and the treatment group jointly applied peer education based on WeChat support. Followed up for 2 months, the two groups of patients were evaluated the degree of social support, psychological flexibility, and rehabilitation self-efficacy by Social Support Rating Scale, Connor-Davidson Resilience Scale, General Self-Efficacy Scale and compared.Results:There was no significant difference in the degree of social support, psychological flexibility, and rehabilitation self-efficacy before intervention between the two groups( P>0.05). The objective support, support utilization, and social support scores in the treatment group after intervention were (9.12±1.12), (10.45 ± 0.75), (32.49 ± 4.56) points, and the control group were (7.45 ± 1.36), (8.74 ± 1.43), (29.84 ± 4.45) points, and the differences were statistically significant ( t values were 6.091, 6.681, 2.658, P<0.01). The scores of toughness, self-improvement, optimism, and mental resilience in the treatment group after intervention were (28.21 ± 4.25), (20.32 ± 3.54), (9.36 ± 1.12), and (57.89 ± 7.21) points, and the control group were (24.36 ± 4.34), (17.14 ± 3.21), (7.84 ± 1.23), (49.34 ± 6.55) points, and the differences were statistically significant( t values were 4.056-5.857, P<0.01). The scores of physical exercise self-efficacy, coping self-efficacy, and rehabilitation self-efficacy in the treatment group after intervention were (43.43 ± 5.38), (54.45 ± 6.32), (97.88 ± 7.45) points, and the control group were (37.45 ± 5.42), (48.65 ± 6.45), (86.10 ± 9.12) points, and the differences were statistically significant ( t values were 5.009, 4.110, 6.430, P<0.01). Conclusions:Peer education based on WeChat support helps to enhance the degree of social support for patients after laparoscopic prostate cancer surgery, improve the level of mental flexibility, and promote the development of rehabilitation self-efficacy.
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Objective@#To detect the expression level of early and late protein of vaccinia virus and to preliminarily explore replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan.@*Methods@#We constructed prokaryotic expression vector, expressed and purified homologous early protein E3 and late protein A27 closely related to replication and prepared mouse polyclonal antiserum by immunizing mice with homologous proteins. Early and late protein expression levels of NTV were detected.@*Results@#We have expressed and purified vaccinia virus proteins respectively in E. coli expression system and prepared homologous mouse polyclonal antiserum. Early protein E3 and late protein A27 could be highly efficient expression in NTV infected non-permissive Hela cells, while expression of late protein F17 was blocked detected by Western blot.@*Conclusions@#The expression limitation of late protein F17 may be an explanation for the replication-defective mechanism of NTV.
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Objective@#To investigate the cellular and humoral immune responses induced by combined immunization with the fusion protein of human papillomavirus type 18 (HPV18) and the recombinant vaccinia virus.@*Methods@#Purified HPV18L231-600E7E6 fusion protein, expressed by prokaryotic expression system, were immunized in combination with the recombinant vaccinia virus vaccine expressing HPV18E7E6 fusion protein (rVV18E7E6) by using various prime-boost regiments in C57BL/6 mice. Cellular and humoral immune responses were analyzed by enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and pseudovirus neutralization assay.@*Results@#Higher levels of cellular immune responses were induced in mice primed with the HPV18L231-600E7E6 fusion protein/adjuvant CpG and boosted with the recombinant vaccinia virus rVV18E7E6, than in other immunized mice. Higher binding antibody level was induced, and low level neutralizing antibody against pseudovirus was detected simultaneously.@*Conclusions@#Priming with HPV18L231-600E7E6 fusion protein/CpG and boosting with the recombinant vaccinia virus rVV18E7E6 could induce higher cellular and humoral immune response in immunized mice, which might be taken as vaccine candidate for treatment of HPV18 chronic infection and postoperative adjuvant treatment for cervical cancer.
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AIM To study the reproductive toxicity of Triptolide,one of the main components of Tripterygium wilfordii Hook.f,on male Caenorhabditis elegans,and its potential mechanism.METHODS Expose L4 larva of him-5 nematodes to 48 h 0.02,0.2,2.0 mg/L of Triptolide.The effect of Triptolide on the fertility of male C.elegans was assessed by crossing test,and the relevant mechanism was explored through measuring spermatids morphology development,spermatids activation,sperm motility and the relative mRNA expression levels of the related spermatogenesis genes.RESULTS Compared with the solvent control group,the 48 h Triptolide treatment induced significantly decreased brood size in 2.0 mg/L Triptolide group,significant reduction in spermatid diameter,spermatid cross-sectional area,rates of spermatid activation,rates of motile sperm in 0.2 and 2.0 mg/L Triptolide groups,and decreased expression levels of spe-10 in 0.02-2.0 mg/L Triptolide groups,decreased spe-15 in 0.2-2.0 mg/LTriptolide groups,and decreased fer-1 and folt-1 in 2.0 mg/L Triptolide groups as well.CONCLUSION Triptolide decreases expression levels of the related genes in spermatogenesis,contributes to an array of indicators inhibition,the spermatids morphology development,the sperm morphology and function development,which leads to decreased fertility of male C.elegans.Therefore,spermatogenesis damage is one of the main pathway that Triptolide adversely affects the fertility.
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Objective@#To generate monkeypox virus specific monoclonal antibodies for further establishing monkeypox virus immunofluorescence assay.@*Methods@#Monkeypox virus A29 protein, vaccinia ortholog A27 protein and cowpox ortholog 162 protein were expressed in E. coli BL21 to screen antibodies. Synthetic monkeypox virus A2917 ~ 49 polypeptide was used to immune BALB/c mice. Monkeypox virus monoclonal antibodies were generated through fusion, cloning and screening techniques. Indirect ELISA was performed to test antibodies specificity and subtype.@*Results@#A29, A27 and 162 proteins were highly expressed in E. coli and detected by Western blot. The three his-tagged proteins were purified using His-Bind affinity chromatography column. The purity of the proteins was all more than 90%. And 8 strains monkeypox virus specific monoclonal antibodies were screened by the three purified proteins. Two mAbs of 8 were IgG3 subtype and the rest were IgG1 subtype.@*Conclusions@#Eight strains of monkeypox virus specific monoclonal antibodies were generated, they can be used to further establish monkeypox virus immune immunofluorescence assay.
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Objective@#To prepare strains of influenza A (H7N9) pseudovirus derived from different districts of China for vaccine efficacy evaluation.@*Methods@#Phylogenetic tree was built based on hemagglutinin (HA) amino acid sequence analyses from 29 influenza A (H7N9) virus strains and 6 influenza A (H7N9) virus strains with HA determinants variation were selected. 293FT cells were co-transfected with plasmid pNL4-3-Luc.R-E-, pVRC-HA and pVRC-NA with codon-optimized hemagglutinin (HA) and neuraminidase (NA) derived from the six influenza A (H7N9) virus strains, respectively. Transmission electron microscopy assay and Western blot analysis were performed to demonstrate morphology and specificity of these particles, luciferase activity assay and hemagglutinin titers detection were used to determine their infectivity and hemagglutinin activity. And finally, pseudovirus-based neutralization assays were evaluated with HA immunized mice serum.@*Results@#Six influenza A (H7N9) peseudovirus particles derived from different districts of China were selected and prepared. All of the particles bearing HA and NA were characterized with classic influenza virus morphology, with TCID50 titer ranged from 104TCID50/50 μl to 105TCID50/50 μl and with hemagglutinin activity ranged from 64 to 512. Neutralization efficacies on influenza A/Shanghai/1/2013(H7N9) HA vaccine serum against 100TCID50 dose of these pseudovirus particles indicated their potential application in the vaccine cross-protective evaluation in future.@*Conclusions@#Six influenza A (H7N9) pseudovirus derived from different districts of China with potential antigenic variation on HA were constructed successfully, established foundation for their further application in vaccine cross-reactive efficacy evaluation.
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Objective To compare the value of shear wave elastography (SWE)and mammography in differential diagnosis of benign and malignant breast neoplasms.Methods Totally 96 patients with breast tumor were randomly chosen and underwent SWE andmammography.The elastic maximum value (Emax)was obtained. Taking histological diagnosis as the golden standards,we compared the two techniques' sensitivity,specificity, accuracy,positive and negative predictive value in diagnosis of breast tumors.Results The sensitivity,specificity, accuracy,positive and negative predictive value of Emax were 91.3%,94.0%,92.7%,93.3% and 92.1%, respectively.The sensitivity and accuracy of E-max were significantly better than those of x-ray (both P <0.05 ). The area under the ROC curve of Emax was 0.983 (95% CI,0.963 to 0.998).Conclusion SWE outperforms mammography in differential diagnosis of benign and malignant breast tumors.
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Objective To compare the value of shear wave elastography (SWE)and mammography in differential diagnosis of benign and malignant breast neoplasms.Methods Totally 96 patients with breast tumor were randomly chosen and underwent SWE andmammography.The elastic maximum value (Emax)was obtained. Taking histological diagnosis as the golden standards,we compared the two techniques' sensitivity,specificity, accuracy,positive and negative predictive value in diagnosis of breast tumors.Results The sensitivity,specificity, accuracy,positive and negative predictive value of Emax were 91.3%,94.0%,92.7%,93.3% and 92.1%, respectively.The sensitivity and accuracy of E-max were significantly better than those of x-ray (both P <0.05 ). The area under the ROC curve of Emax was 0.983 (95% CI,0.963 to 0.998).Conclusion SWE outperforms mammography in differential diagnosis of benign and malignant breast tumors.
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To compare different adjuvant formulation and explore the impact of Calcineurin B subunit(CnB) as adjuvant with a novel HBV protein particle (HBSS1) vaccine in mice, female C57BL/6 mice were immunized HBSS1 with Al(OH)3 only, or a normal dose (5 μg) CnB only, or (CnB+ Al(OH)3) mixture as the adjuvant. All immunized groups were primed twice at 4-week intervals; followed by boosting with recombinant adenoviral based HBV vaccine(rAdSS1) at 10-week intervals. We detected the antigen specific humoral response in mice, including total IgG antibody and IgG subtyping. Then, we characterized the specific cell-mediated immune (CMI) response by detection of γ-interferon secreting splenocytes after stimulaton with S or PreS1 peptide pools. No enhancement of immunity was found among the mice with 5 μg of CnB alone or combined with Al(OH), adjuvanted vaccine,which could not induce higher level of anti-PreS1 and anti-S antibodies and CMI than that of HBSS1 alone or Al(OH)3 adjuvanted vaccines. We concluded that CnB is not an effective adjuvant for a novel HBV subunit vaccine.
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Animals , Female , Mice , Adjuvants, Immunologic , Pharmacology , Calcineurin , Pharmacology , Hepatitis B Vaccines , Allergy and Immunology , Mice, Inbred C57BL , Protein SubunitsABSTRACT
<p><b>OBJECTIVE</b>To map the frequency and types of EGFR gene mutations present in lung cancer tissues. To evaluate the clinical applicability of a novel real-time double-loop probe PCR of which the ADx-EGFR kit is based, and to compare its performance with traditional Sanger DNA sequencing in the detection of somatic mutations of tumor genes.</p><p><b>METHODS</b>A total of 208 formalin-fixed paraffin-embedded (FFPE) tumor samples were tested. Genomic DNA of the tissue samples was extracted and purified, and subjected to both traditional PCR amplification, Sanger sequencing of EGFR gene in exon 18, 19, 20, 21, and ADx's EGFR mutation detection kit. The mutation rates for EGFR gene in exon 18, 19, 20, 21, as well as the frequency of each mutation detected by the two methods, were analyzed.</p><p><b>RESULTS</b>The traditional Sanger DNA sequencing technique was successfully performed in 196 out of 208 (94.2%) lung cancer samples, and 22 samples (11.2%) showed EGFR gene mutations. ADx-EGFR kit was successfully used in the lung cancers of all of the 208 cases (100.0%), and 40 samples (19.2%) showed mutations. In the lung cancer samples analyzed, mutations were mainly detected in the exon 19 and exon 21 L858R point mutation, i.e. 4.8% (10/208) and 11.6% (23/208) of total mutations, respectively, and the remaining mutations were rare.</p><p><b>CONCLUSIONS</b>The success rate of ADx-EGFR real-time PCR for formalin-fixed and paraffin-embedded tissues samples is significantly higher than that of Sanger sequencing (P < 0.01). There are significant differences between the two methods. ADx-EGFR real-time PCR shows a much higher successful detection rate and mutation rate of lung cancer tissues compared with that of Sanger sequencing. As a result, the real-time PCR with ADx-EGFR kit is proved to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is an effective and reliable tool for clinical screening of somatic gene mutations in tumors.</p>
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Humans , DNA Mutational Analysis , Methods , Exons , Genes, erbB-1 , Lung Neoplasms , Genetics , Paraffin Embedding , Point Mutation , Real-Time Polymerase Chain Reaction , MethodsABSTRACT
<p><b>OBJECTIVE</b>To express HPV31 and 52 L2 fusion protein and detect its immunogenicity.</p><p><b>METHODS</b>According to the amino acid sequences of HPV31 and 52 L2 11-200AA published in the GenBank database, weartificially synthesized the HPV31 and 52 L2 fusion gene which was optimized according to Escherichia coli codon usage and encodes 11-200 amino acid of HPV31 and HPV52 L2, then cloned it into pET-9a vector. The HPV31 and 52 L2 fusion protein was expressed in Prokaryotic expression system and the mice were immunized with the fusion protein after purification. The immunogenicity was characterized in vaccinated mice.</p><p><b>RESULTS</b>HPV31 and 52 L2 fusion protein was highly expressed in E. coli, the amount of fusion protein is nearly 20% of the total bacterial protein. The purified fusion protein with aluminum adjuvant could induce specific high titer of IgG antibodies detected by ELISA, and also induce the neutralizing antibodies against pseudovirus of HPV31 and HPV52 and cross-neutralizing antibodies against pseudovirus of HPV45, 58, 16, 18.</p><p><b>CONCLUSION</b>HPV31 and 52 L2 fusion protein could induce neutralizing and cross-neutralizing antibodies against HPV pseudovirus. It provides laboratory basis for development of HPV L2 protein vaccine.</p>
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Animals , Female , Mice , Antibodies, Viral , Blood , Escherichia coli , Genetics , Mice, Inbred BALB C , Oncogene Proteins, Viral , Genetics , Allergy and Immunology , Papillomaviridae , Allergy and Immunology , Papillomavirus Vaccines , Allergy and Immunology , Recombinant Fusion Proteins , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To determine the antigen characteristics of different fragments of SARS-CoV N protein expressed in E. Coli and their application in the serological diagnosis.</p><p><b>METHODS</b>Based on preliminary analysis of 39 different segments of the N protein, We choosed six purified N protein for further antigenicity characterization in this study, including that PN360 (1 -360aa), PN301 (1-301aa), PN199 (30-228aa), PN185 (30-214aa), PN155b (60-214aa), and PN125 (90-214aa). We developed Western-Bolt and ELISA to detect antibody reactivity between truncated N fragments with sera from SARS-CoV-negative normal adults or SARS-CoV patient convalescent sera.</p><p><b>RESULTS</b>Western-Bolt results show that all the six fragments have reacted with the SARS patient convalescent sera, but the PN360 and PN301 showed obvious cross-reaction with sera from SARS-CoV-negative normal adults; sensitivity analysis using an ELISA coating with PN199, PN185, PN155b, PN125 as antigen showed that the PN185 and PN155b are better than PN125.</p><p><b>CONCLUSION</b>Truncated N protein PN185 and PN155b expressed in E. Coli are better antigen candidates used for detection of SARS-CoV specific antibody.</p>
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Humans , Antibodies, Viral , Blood , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Genetics , Nucleocapsid Proteins , Allergy and Immunology , Peptide Fragments , Allergy and Immunology , Recombinant Proteins , Allergy and Immunology , Serologic Tests , Severe Acute Respiratory Syndrome , DiagnosisABSTRACT
<p><b>OBJECTIVE</b>To investigate the high expression of HPV16L2N120E7E6 fusion protein by prokaryotic expression system, and evaluate its immunogenicity and antitumor efficacy in vaccinated mice.</p><p><b>METHODS</b>The HPV16L2N120E7E6 fusion gene, its codons were optimized to increase the expression of the protein, was constructed by overlap extension PCR and inserted into prokaryotic expression vector pET9a. Then the fusion protein was expressed by inducing with IPTG in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and further detected by SDS-PAGE and Western-blot. Finally, the humoral and cellular immune responses were measured by ELISA and ELISPOT, respectively, in vaccinated mice with the purified HPV16L2N120E7E6 fusion protein, and the antitumor efficacy was assessed in mice using the TC-1 tumor challenge model.</p><p><b>RESULTS</b>The codon-optimized HPV16L2N120E7E6 fusion gene was highly expressed in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and the amount of fusion protein was nearly 48.6% of the total bacterial protein. The purified fusion protein could induce high titer of specific antibody against L2, E7 and E6 in vaccinated mice. When accompanied with the adjuvant CpG, the fusion protein was able to elicit strong and moderate cellular immune responses in vaccinated mice against peptide HPV16E7(49-57) and peptide pools of HPV16E6, respectively. Furthermore, the tumor therapeutic experiment showed that HPV16L2N120E7E6 + CpG could prevent the tumor formation in 80.0% (8/10) vaccinated mice.</p><p><b>CONCLUSIONS</b>The data of this study suggest that HPV16L2N120E7E6 fusion protein could be a promising candidate vaccine for treatment of chronic HPV16 infection and post-operative adjuvant therapy for cervical cancer.</p>
Subject(s)
Animals , Female , Humans , Mice , Adjuvants, Immunologic , Pharmacology , Cancer Vaccines , Allergy and Immunology , Therapeutic Uses , Capsid Proteins , Genetics , Allergy and Immunology , Metabolism , Cell Line, Tumor , Cell Proliferation , Codon , Escherichia coli , Allergy and Immunology , Metabolism , Immunization , Methods , Immunotherapy , Methods , Mice, Inbred C57BL , Neoplasm Transplantation , Oligodeoxyribonucleotides , Allergy and Immunology , Oncogene Proteins, Viral , Genetics , Allergy and Immunology , Metabolism , Papillomavirus E7 Proteins , Genetics , Allergy and Immunology , Metabolism , Papillomavirus Vaccines , Allergy and Immunology , Therapeutic Uses , Plasmids , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Metabolism , Repressor Proteins , Genetics , Allergy and Immunology , MetabolismABSTRACT
Objective To investigate the influence of indomethacin to the unite treatment effect on chronic phase myeloid leukemia (CML-CP) with interferon alpha-2b (IFNα-2b) and low dose cytarabine (LD-Ara-C).Methods 22 CML-CP patients were randomly divided into two groups.Control groups (10 cases) injected with IFNα-2b (3 million units),injection frequency was q.o.d,the duration of treatment was about 3-18 months,cytarabine (Ara-C) by slowly intravenous driped (30 mg/d).In this treatment schedule,every course of treatment sustained 10 days,and with a 2 weeks interval.During this process,patients in treatment group were treated with hydroxyl urea only when their WBC in peripheral blood exceed 20×109/L,otherwise,discontinue it.Treatment group (12 cases),on the first day of treatment with IFNα-2b and Ara-C,added indomethacin (25 mg) through oral administration,the frequency was t.i.d.During treatment in the two group,the end point of observation was completely hematology ease,at the same time,these indicators in the two groups needed to be compared,the time when WBC begin to fall,the time when WBC fall to normal range,the time when immature cells returned to normal,the time which complete hematological remission and the highest temperature of patients after IFNα-2b was subcutaneous injected.Results The time when WBC begin to fall in treatment group was (4.2±2.7) d,and the time was (5.0±2.5) d in control group (t =0.714,P ＞ 0.05).In treatment group,the time when WBC fall to normal range was(10.0±4.5) d,and the time was (12.0±4.5) d in control group (t =1.036,P ＞ 0.05).The time when immature cells returned to normal in treatment group was (14.2±4.8) d,and the time was (19.0±3.6) d in control group (t =2.609,P ＜ 0.02).The time which complete hematological remission was achieved in control group was (45.8±5.6) d,but it was (53.9±10.5) d in control group (t =2.314,· P ＜ 0.05).Meanwhile,the fever degree after IFNα-2b was subcutaneous injected obviously achieved improvement in treatment group (x2 =12.041,P ＜ 0.005).Conclusion The advantage of indomethacin to the unite treatment with IFNα-2b and LD-Ara-C on CML mainly lays in which cound alleviated the adverse reaction such as flu-like of IFN,and more,there are synergy effect in antagonist CML.
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ObjectiveTo construct one recombinant adenovirus AdE7E6 expressing HPV16 E6 and E7 fusion protein as candidate for HPV16 therapeutic vaccine.MethodsThe codon-optimized E6 and E7 gene,were fused to create one open reading frame,then inserted into adenovirus vector pCD316.A strain of recombinant adenovirus was constructed through homologous recombinant in 293 cells,and identified by PCR and Western blot.Finally,it was employed to study it's immunogenicity and the activity of the tumor growth regression.ResultsThe PCR result showed that E6E7 fusion gene had been integrated in recombinant Ad5 DNA.Western blot test confirmed that the E6E7 fusion protein was highly expressed in 293 cells infected with Ad5E7E6 recombinant adenovirus.The recombinant adenovirus elicited significant E7 specific CD8+ T lymphocyte response in vaccinated mice.These responses could completely prevent the TG-1 tumor cell bearing mice treated with AdE7E6 from developing into tumor.ConclusionThese results suggested that rAd5E7E6 could be a potent vaccine candidate for the treatment of HPV16-associated tumors and their precancerous transformations.
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To investigate the seasonal influenza split vaccine's immune protective effectiveness against the homologous and heterogonous subtypes of influenza A virus challenge and the relationship between the protective effectiveness and hemagglutination inhibition (HI) antibody titer in mice. Two components of H1N1 and H3N2 in Chinese 2008-2009 seasonal influenza spilt vaccine, were derived from vaccine strain A/Brisbane/59/2007 (H1N1)-like virus and A/Brisbane/10/2007 (H3N2)-like virus respectively, and were used to immune BALB/c mice. Firstly, different doses of the vaccines were used to immunize mice and the HA immunization dosage that can induce the HI antibody titer of 40 in mice was identified; Secondly, H1N1 vaccine immunized mice were challenged with different doses of influenza virus mouse adaptation strains of A/Brisbane/59/2007 (H1N1)-like virus (MA) (referred to as A1 virus, well matched-strain in the homologous subtype) and A/Purto Rico/8/34 (H1N1) (referred to as PR8 virus, poor matched-strain in the homologous subtype) respectively, and H3N2 vaccine immunized mice were challenged with H1N1 influenza virus of A1 strain (Heterogonous subtype), body weight changes and survival rates were observed to explore the immune protective effectiveness of influenza split vaccine against the homologous and heterogonous subtypes of influenza A virus in mice. Results indicated that HI antibody titers were elevated as the HA protein immunization dosages increased from 0.15 microg, 0.5 microg, 1.5 microg, 5 microg to 15 microg in mice, and 1.5 microg HA of the seasonal influenza split vaccine could induced HI antibody titer of 40 in mice; 3LD50, 10LD50, 30LD50, 100LD50, 300LD50,1000LD50 and 3000LD50 of influenza virus strain A1 were used to challenge the H1N1 immunization mice, 1.5 microg HA of H1N1 vaccine could 100% protect mice against challenge with 1000LD50 of matched and homologous subtype of influenza virus strains A1, mice immunized with 15 microg HA of H1N1 vaccine even could 100% protect mice against challenge with 3000LD50 of influenza virus strains A1; but mice immunized with both the 1.5 microg and 15 microg HA of H1N1 vaccine were all sacrificed when challenged with 3LD50 of the mismatched and homologous subtype of influenza virus strain PR8, and mice immunized with the high dosage of 15 microg HA of H3N2 vaccine also were all sacrificed when challenged with 3LD50 of the heterogonous subtype of influenza virus strain A1. These results suggest that 1.5 microg HA of seasonal influenza split vaccine could induced HI antibody titer of 40 after one dose in mice, this dosage of HA can effectively protect mice against matched homologous subtype of influenza virus strain, but hardly to protect mice against mismatched homologous or heterogonous subtype of influenza virus strain. These results provide materials for the establishment of influenza vaccine evaluation system based on seasonal influenza vaccine.
Subject(s)
Animals , Chick Embryo , Dogs , Female , Mice , Antibodies, Viral , Blood , Cells, Cultured , Influenza A Virus, H1N1 Subtype , Allergy and Immunology , Influenza Vaccines , Allergy and Immunology , Mice, Inbred BALB C , Orthomyxoviridae Infections , VaccinationABSTRACT
This investigation is to delete the most of the coding sequence (1104 bp) of the IV a2 gene in an adenovirus genome by a lambda-Red recombinase system-mediated PCR-targeting approach and rescue a recombinant adenovirus with IV a2 deletion. First, the template pAK of PCR targeting, containing kanamycin cassette, was constructed. Then, a linear fragment for PCR targeting, which had 39 bp homologous arms at both of its terminus, was amplified by PCR from the pAK. The pFG140 and the linear fragment were electroporated into E. coli BW25113/pIJ790 sequentially and the recombinant pFG140-deltaIV a2 (1104) was established by homologous recombination between the linear fragment and the pFG140 with aid of X-Red recombinase. The precise deletion of 1 104 bp fragment from IV a2 was confirmed by restriction endonucleases digestion and DNA sequencing. ORF of IV a2 was amplified by PCR from pFG140 and then cloned into the pAAV2neo vector. The recombinant adenovirus Ad5delta IV a2 (1104) was rescued by co-transfection of pFG140-deltaIV a2 (1104) and pAAV2neo-IV a2 into HEK293 cells. It was shown by Western Blot that IV a2 could not be detected in the Ad5deltaIV a2 (1104)- infected HEK293 cells. This study established a PCR-targeting strategy for manipulating adenovirus genome directly by a lambda-Red recombinase system, and a recombinant adenovirus with IV a2 deletion was obtained.
Subject(s)
Humans , Adenoviridae , Genetics , Genome, Viral , HEK293 Cells , Polymerase Chain Reaction , Recombinases , Metabolism , Sequence Deletion , Viral Proteins , Genetics , Virus AssemblyABSTRACT
To investigate the genetic stability (including the vector of vaccinia virus and six foreign genes: gp160, gag, pol, rev, tat and nef) of the HIV-1 non-replicating recombinant vaccinia virus (rNTV-C). rNTV-C was serially passaged to passage 25 (P25) in primary chicken embryo fibroblast (CEF). P9, P12, P15 and P25 were selected to study the genetic stability in four aspects, including the genetic stability of viral vector, the genetic stability of six foreign genes, the expressing stability of foreign genes and the genetic loss of foreign genes. The results showed that the viral vector was non-replicated vaccinia virus of Tiantan strain and was passaged stably; foreign gene sequences matched with designed sequences, the insert sites were right, and the nucleotide mutation rate was less than one over ten thousands within different passages of rNTV-C; the target proteins could be expressed effectively, and the expression level was stable within different passages of rNTV-C; the genetic loss of gag and nef was less than 5% within different passages of rNTV-C. The above results provided important data for the vaccine production.
Subject(s)
Animals , DNA, Recombinant , Genetics , Fibroblasts , Metabolism , Virology , Gene Expression , Genes, Viral , Genetics , Genetic Engineering , Methods , Genetic Vectors , Genetics , HIV-1 , Genetics , Sequence Analysis, DNA , Vaccinia virus , GeneticsABSTRACT
To efficiently express nucleoprotein (NP) of influenza A virus A/Jingke/30/95 (H3N2) in E. coli for further immunogenicity study, three forms of NP gene, NP(His) (NP fused with 6 x His tag), NPwt (wild type NP, non-fused NP with native codon) and NP(O) (codon optimized, non-fused NP) were cloned by the technologies of restriction enzyme digestion, PCR, codon optimization and gene synthesis. Three recombinant plasmids were subsequently constructed based on the prokaryotic vector pET-30a, respectively. The comparative studies with these plasmids were carried out on the gene expression efficiency, induction temperature and time, purification process and immune reactivity. It was confirmed by restriction enzyme digestion and sequencing analysis that the three NP genes were inserted into the expression plasmid pET-30a correctly. SDS-PAGE showed that all three forms of NP gene could be efficiently ex pressed in E. coli, among which NP(O) was expressed with the highest expression level. The lower temperature fermentation (T=25 degrees C) and longer time induction (t=10 h) were necessary for high-level expression of protein in soluble form. The purity of tag-free NP was up to 90% through the two-step purification process with anion-exchange and gel filtration chromatography. It was indicated by Western blot that purified NP reacted well with the serum from mice immunized with PR8 virus. These results suggest that the codon-optimized influenza A virus NP gene can be efficiently expressed in E. coli and the expressed NP protein with specific immune reactivity could be purified from the supernatant of bacterial lysate.