ABSTRACT
Objective To develop, optimize and preliminarily verify an indirect immunofluores-cence assay ( IFA) for detecting the titer of recombinant baculovirus. Methods Conditions for performing IFA, including cell concentration, co-incubation time, reaction temperature, dilution ratio, reaction time and types of fixative solution, blocking liquid and antibodies, were optimized to establish an IFA method for the detection of baculovirus titer. Specificity, accuracy, reproducibility and intermediate precision of the es-tablished assay were verified. And the results were compared with those of baculovirus rapid titer kit. Re-sults The optimal cell concentration for coating was 0. 6×106 cell/ml, and the optimal reaction time be-tween viruses with cells was 3 d. The optimal conditions for conducting IFA were as follows: formaldehyde buffered acetone (-20℃) was used as fixative, normal goat serum was used as blocking liquid and the first and second generation antibodies at a dilution of 1 : 200 were incubated at 37℃ for 1 h, respectively. Spe-cific fluorescence was observed only in baculovirus but not in others by using the method. No significant difference in virus titers was observed between the established IFA and baculovirus rapid titer kit. The two methods showed a good linear correlation (R2=0. 996). Coefficients of variation for evaluating the reproduc-ibility and intermediate precision were less than 10％. Conclusion IFA for the detection of baculovirus ti-ter was established with good specificity, accuracy, reproducibility and intermediate precision.
ABSTRACT
Objective:To prepare and screen monoclonal antibodies against Herpes simplex virus-1(HSV-1),and develop a double antibody sandwich quantitative enzyme-linked immunosorbent assay( Q-ELISA) for detection of HSV-1 particle. This method was used to control the quality of viral particle in the developing and manufacturing process of HSV-1. Methods: BALB/c mice was immunized with HSV-1 to prepare monoclonal antibodies. A double antibody sandwich Q-ELISA was developed to determine concentration of HSV-1 particle,which was based on the neutralizing monoclonal antibody 1F6 as capture antibody,and 2B1 as HRP-conjugated antibody. The performance of the reagent was evaluated,including specificity,sensitivity,precision,accuracy and linear. And the relation between the amount of virus detected by this method and the virus titer was analyzed by regression analysis method. Results: The Q-ELISA for HSV-1 particle was developed. The quantitation scope was 0. 125-2 μg/ml, the coefficient correlation was 0. 995 5, the limit of detection was 0. 125 μg/ml, the recovery was between 85. 6% and 107. 1%, the variation coefficient was lower than 10%, and the reagent does not react with other samples except HSV-1 antigen. This method has a good correlation with virus titer. Conclusion:The Q-ELISA for HSV-1 particle was successfully developed,which provide a new approach for rapid and quantitative detection of HSV-1 antigen.
ABSTRACT
Objective To establish the crystal violet plaque assay for detection of virus titer of recombined Tiantan vaccinia AIDS vaccine,and provide more stable method of virus titration for rTV AIDS Vaccine.Methods Optimized the concentration of Vero cells,the time and temperature of virus adsorption,and the time of determination for CPE,then established the crystal violet plaque assay for virus titer of rTV.Counting and analysis the plaques by BioSpot Reader,then analyzed the relativity of plaques counted with BioSpot Reader and manual; Several lots rTV AIDS Vaccine and Tiantan vaccinia were titrated by the method of plaque formation-hemadsorption assay,neutral red and crystal violet plaque assay,then analyzed the relativity of the results of three methods ; meanwhile,the virus titer of samples were determine repeatedly by the crystal violet plaque assay,then calculated the coefficient of variation( CV),and verified the precision of the method; SPSS17.0 was used in statistical analysis of the experimental results.Results When the concentration of Vero cells was 5.0×105-9.O×105 cells/ml,virus been adsorbesd 2 h at 37℃,then cultivated 72 h after adding the culture medium containing methyl cellulose.Plaques counted by BioSpot Reader was highly related with counted by manual (r =0.985),so BioSpot Reader counting can objectively reflect the virus plaques with various size,and reduce the error by manual counting; compared the virus titration for different lots of rTV AIDS vaccine and Tiantan vaccinia with three methods,the crystal violet plaque assay was highly related with plaque formation-hemadsorption assay (r =0.997,P＜0.01 ) and neutral red plaque assay(r=0.980,P＜0.01 ).Conclusion Crystal violet plaque assay was established for virus titration of rTV AIDS Vaccine.
ABSTRACT
Objective To ensure the quality of live attenuated measles vaccine on the market,viral titers were determined as the market surveillance test.Methods The measles vaccine was sampled from sites located in 24 provinces (cities,autonomous regions) of China,including provincial CDC,municipal CDC,county CDC,and primary users.The virus titer of samples was tested to evaluate the quality status of measles vaccine on the market.Results For all the 54 lots of measles vaccine sampled in this study,the virus titers were in compliance with the requirements of the China Pharmacopeia Vol.III (2005 edition).In comparison with the original test results provided by the manufacturers,the mean virus titer was decreased 0.2 lg?0.3.Conclusion Viral titers of measles vaccine batches sampled from the market were all in compliance with the requirements of the China Pharmacopeia Vol.III (2005 edition).It showed that the measles vaccine in China market has good stability,and the cold chain system for measles vaccine conveyance and storage could ensure the vaccine efficacy.
ABSTRACT
AIM: To develop a real - time PCR assay based on TaqMan technology for the identification of recombinant baculovirus and determination of virus physical titers in Bac - to - Bac system. METHODS: The recombinant baculovirus containing human IL- 18 gene was produced using Bac -to- Bac system. A 10 -fold serially diluted primary viral stock was used for plaque assay and DNA extraction. Bacmid (baculovirus plasmid) was 10 -fold serially diluted and served as standards. Real - time PCR amplification of the IL - 18 gene was performed in triplicate for each diluted recombinant virus. At the same time, plaque assays were performed using overlay agarose method. RESULTS: The standard linear range (101 to 108 copies) for quantitation was achieved with the standard curve. We also find that the"vg/mL"titer value is generally about 10 times than"pfu/mL"titer of the same recombinant virus stock. CONCLUSION: A TaqMan real -time PCR method is established to identify the recombinant baculovirus and determine the"vg/mL"titer of virus. The method is rapid and quantitative over a wide range of virus titers.
ABSTRACT
To study the contribution of T cell subsets in the pathogenesis of Murine hepatitis virus Type3 (MHV-3) induced chronic viral hepatitis in C3H/Hej mice, ninety C3H/Hej mice were chosen to individually receive 10 plaque forming units (PFU) of MHV-3 intraperitoneally. The changes of virus titer and pathology in liver tissue were examined by standard plaque assay and by the hematoxylin/eosin (HE) staining method from 2 days post MHV-3 infection. The ratios of T cell subsets including CD3+CD4+CD8-, CD3+CD4-CD8+, CD3+CD4-CD8-, CD3+CD4+CD25+, CD3+CD4+CD25- and CD3+CD4-CD25+ T lymphocyte of total T lymphocytes in blood, spleen and liver were examined at 0, 2, 4, 6,8, 10, 12, 15, 20, 25, 30, 40 days post MHV-3 infection by flow cytosorting. We observed that the virus titer raised and showed persistent virus duplications and inflammatory changes in the livers of C3H/Hej mice from 2 days post MHV-3 infection. The double negative T cell (DN Treg cell) and CD4+CD25+ T cell ratios increased significantly from 2 days post MHV-3 infection in C3H/Hej mice, and CD3+CD4+CD8-, CD3+CD4-CD8+, CD3+CD4+CD25- and CD3+CD4-CD25+ T cell ratios decreased accordingly. In conclusion, the changes of virus titer and pathology in the livers of C3H/Hej mice post MHV-3 suggest their contribution to viral persistence. Further characterizations of DN Treg cells are that infection indicates that MHV-3 could induce the chronic inflammation in livers of C3H/Hej mice.The increase of the DN Treg cell and CD4+CD25+ T cell ratios in C3H/Hej mice post MHV-3 infection suggests that DN Treg cells and CD4+CD25+ T cells may both have important suppressive immunomodulation functions in the development of chronic viral hepatitis and have important roles in the virus persistent infection. Further characterizations of DNT cell and CD4+CD25+ T cell are under investigation.
ABSTRACT
Objective To explore the effect and mechanism of Liq Qibei against herpes simplex virus-Ⅱ(HSV-Ⅱ) for treatment of cervical erosion.Methods Using micro-inhibition cytopathic and in vitro cell culture technique,the various concentrations of Liq Qibei in inhibiting HSV-Ⅱ proliferation and the direct virucidal effect were studied.The virus titer and virus inhibition index were taken as parameters.Results ① Virus proliferation experiment:Compared with control group,the virus titer of 1.63,0.81,0.41 mg/mL Liq Qibei group had significant difference(P2.② Direct virucidal effect of Liq Qibei:comparing with control group,1.63 mg/mL Liq Qibei in 8 h,24 h and 0.81 mg/mL-24 h groups had marked difference(P0.05).Conclusion Liq Qibei antivirus effect on HSV-Ⅱ.
ABSTRACT
Retroviral expression system which consists of retroviral vector,envelop protein vector and packaging cell line is an efficient expression system for recombinant protein.It has great potential in gene therapy and biopharmacy.Transcriptional active genome regions are the preferred targets for retrovirus integration.Furthermore,VSV-G protein enables this system a broader host range and makes virus integration more efficient.After infection of high-titer virus,high production clones can be selected through simple screening.So far,the research on retrovirus expression system has developed into application in bio-pharmacy industry.Here the composition of this system and the mechanism of virus transduction and summarize the application and prospect of retroviral expression system are introduced.
ABSTRACT
Objective:To develop a real-time PCR assays based on TaqMan chemistry for the identification of recombinant baculovirus and determination of virus physical titers in Bac-to-Bac system.Methods:The recombinant baculovirus containing human IL-18 gene was produced using Bac-to-Bac system.A 10-fold series diluted primary viral stocks were used for plaque assay and DNA extraction.Bacmid(baculovirus plasmid) was 10-fold series diluted and served as standards.Real-time PCR amplification of the IL-18 gene was performed in triplicate for each diluted recombinant virus.At the same time,plaque assays were performed using overlay agarose method.Results:The standard linear(101 to 108 copies) from quantitation was achieved with the standard curve.We also find that the "vg/ml" titer value is generally about 10 times than "pfu/ml" titer of the same recombinant virus stock.Conclusion:A TaqMan real-time PCR method is established to identify the recombinant baculovirus and determine the "vg/ml" titer of virus.The method is rapid and quantitative over a wide range of virus titers.
ABSTRACT
Objective To establish a recombinant retroviral vector containing insulin-like growth factor-1(IGF-1) gene and to provide the basis for the application of IGF-1 in treating nervous system diseases such as stroke.Methods The plasmid pcDNA3.1-IGF-1 was cut by EcoR I/Xho I,and subcloned to retroviral vector pLXSN,resulting in the recombinant plasmid(pLXSN-IGF-1.)The recombinant IGF-1 expression vector was evaluated by using enzyme cutting and sequencing.By the Lipofectamine 2000,pLXSN-IGF-1 was transferred to packaging cell line-pA317.Culture supernatant of these cells was detected for titration of the recombinant virus.Results The two fragments from recombined IGF-1 eukaryotic expression vector by EcoR I and Xho I represented 400?bp and 6.0?kb by agarose electrophoresis,and PCR showed positive fragment which was about 400?bp long,and sequence analysis showed the same sequence as expected.The cell line pA317-IGF-1 was established,and average virus titer of the recombinant virus in the culture supernatant was about 6.5?10~5 CFU/ml.Conclusion A recombinant retroviral containing IGF-1 gene was successfully constructed.