ABSTRACT
To find out whether the mutations of HIV-1 Env have influence on the assembly of pseudovirus and their abilities to infect cells, site-directed mutation (A457D)was performed using cycling mutagenesis and selection of mutants with DpnI. Transformation and plasmid purification technologies were used to obtain mutated env clone. Then both the prototype and the mutant were co-transfected with pSG3(delta(env)) to 293FT cells, respectively. Single-cycle infection assay was employed to analyze the effect of the prototype and the mutant on the ability of functional pseudovirus assembly. The transient expression of both the prototype S12-42-1 and mutant S12-42M were confirmed by Western blot essay. The S/CO value was less than 1 for S12-42-1 and 6.65 for S12-42M, demonstrating the functional pseudovirus was generated only for S12-42M. So mutation on HIV-1 Env has influence on the assembly of pseudovirus and their abilities to infect cells.
Subject(s)
Humans , Amino Acid Sequence , Base Sequence , Cell Line , HIV Infections , Virology , HIV-1 , Chemistry , Genetics , Physiology , Molecular Sequence Data , Mutation , Sequence Alignment , Virion , Genetics , Physiology , Virus Assembly , env Gene Products, Human Immunodeficiency Virus , Chemistry , Genetics , MetabolismABSTRACT
In order to evaluate the immunogenicity of HPV 58 L1 DNA vaccines, five DNA vaccines had been constructed with pcDNA3.1 vector containing different L1 genes of HPV 58, which were designated as L1h, L1hDeltac, L1S, L1SM and L1wt. The protein expression of DNA vaccines in vitro was tested by Western blot. The ability of forming pseudovirus was evaluated by transfecting DNA vaccine together with pcDNA3.1-h58L2 and pcDNA3.1-GFP into 293FT cells. The neutralizing antibodies and cellular immune response produced in BALB/c mice immunized with the DNA vaccines were detected by using pseudovirus-based neutralization assay and ELISPOT respectively. The results showed that the five DNA vaccines had been successfully constructed; the level of protein expression of L1hDeltac was the highest and those for L1S and L1SM were of medium, while no expressed target protein of L1wt was detected. Only L1S could form the pseudovirus while the other four vaccines could not. L1S and L1h could induce neutralizing antibody. However, the average titer of neutralizing antibody for L1S (1:6,400) was much higher than that for L1h (1:48) and the other three vaccines could not induce neutralizing antibody. No cellular immune response for all five DNA vaccines was detectable by ELISPOT. The results indicated that DNA vaccine against HPV 58 can form pseudovirus in vitro, also can induce high level of neutralizing antibodies. This provides reference for screening HPV vaccine in future.
Subject(s)
Animals , Female , Humans , Mice , Blotting, Western , Cell Line , Enzyme-Linked Immunosorbent Assay , Green Fluorescent Proteins , Genetics , Metabolism , Immunization , Mice, Inbred BALB C , Models, Genetic , Neutralization Tests , Papillomaviridae , Allergy and Immunology , Papillomavirus Vaccines , Genetics , Allergy and Immunology , Recombinant Fusion Proteins , Genetics , Metabolism , Transfection , Vaccines, DNA , Genetics , Allergy and ImmunologyABSTRACT
A bacterial strain which could grow well on the substrate of PAEs as the sole source of carbon and energy was isolated from contaminated sludge in the river of WeiFang in ShangDong province and it was designated as JDC-3. Based on the morphology,biophysical and biochemical properties as well as molecular characteristics,this isolate was preliminarily identified as Delftia sp.. A fragment of phthalate dioxygenase gene was successfully amplified from the genus of Delftia for the first time using a set of degenerate primers. Meanwhile,the degradation capability of JDC-3 was determined by HPLC using DMP as test substrate. The results showed that the optimal pH and temperature were at 7.0～8.0 and 30?C～35?C respectively. The degradation kinetics of JDC-3 was studied in different initial DMP concentration under optimal conditions. The results indicated that the degradation dynamic equation was ln C =-0.06837 t + A when DMP concentration was lower than 300 mg/L,with half life of 12.48 h. The degradation rate decreased and half life of JDC-3 prolonged as the initial concentration kept on increasing.
ABSTRACT
To have a better understanding on the evolution of Metallosphaera sedula-mediated precipitation and the properties of the precipitate synthesized by this species，a newly identified extremely thermophilic strain (YN23) of Metallosphaera sedula was cultured in the medium containing Fe2+ as energy resouTce under optimal condidons (pH1.5，65℃,0.2g·1-1 yeast extract,30g·1-1 Fe2SO4·7H2O and 170rpm)．XRD，EDS，ETIR and SEM reveal the precipitate obtained from YN23-inoculated flasks to be a mixture of potassium jarosite and ammoniojarosite，with morphological features similar to the latter．Precipitation Was first detected once over 90％ of Fe2+ was oxidized at hour 25 with a peak pH value of 1.92，and was ended when precipitate reached the highest point of 7.9g·1-1 at hour 95 with the lowest pH value of 1.32 in solution．Microbial density underwent a rapid increase along with Fe2+ oxidation and a gradual decrease with precipitate piling up.
ABSTRACT
To have a better understanding on the evolution of Metallosphaera sedula-mediated precipitation and the properties of the precipitate synthesized by this species，a newly identified extremely thermophilic strain (YN23) of Metallosphaera sedula was cultured in the medium containing Fe2+ as energy resouTce under optimal condidons (pH1.5，65℃,0.2g·1-1 yeast extract,30g·1-1 Fe2SO4·7H2O and 170rpm)．XRD，EDS，ETIR and SEM reveal the precipitate obtained from YN23-inoculated flasks to be a mixture of potassium jarosite and ammoniojarosite，with morphological features similar to the latter．Precipitation Was first detected once over 90％ of Fe2+ was oxidized at hour 25 with a peak pH value of 1.92，and was ended when precipitate reached the highest point of 7.9g·1-1 at hour 95 with the lowest pH value of 1.32 in solution．Microbial density underwent a rapid increase along with Fe2+ oxidation and a gradual decrease with precipitate piling up.