This study aims to establish a method for counting the viral particles in adenovirus vector-based vaccines. Nano-flow cytometry was employed to analyze the viral particles in adenovirus-based vector vaccines at the single-particle level. Monodisperse silica nanoparticles with a refractive index close to that of the virus were selected as the particle size standard to calculate the viral particle size, which was then compared with the results obtained from transmission electron microscopy (TEM) to determine the gating strategy. Subsequently, a particle count standard was employed to calculate the viral particle concentration. The established method demonstrated good linearity, accuracy, precision, and specificity. The results of determined viral particle concentration showed a good correlation with the infectious titer. Compared with the conventional OD₂₆₀ method, nano-flow cytometry can directly measure the viral particle concentration and indicate whether the sample has been disassembled according to changes in viral particle concentration and size, thus more accurately reflecting the actual infectious potency of the sample. The novel quantification method established in this study is capable of indicating the efficacy of adenovirus vector-based vaccines and provides effective technical support for the quality control of such products.
Adenoviridae/genetics* ; Genetic Vectors ; Flow Cytometry/methods* ; Virion/isolation & purification* ; Particle Size ; Nanoparticles ; Viral Vaccines

Stomatal density is important for crop yield. In this paper, we studied the epidermal pattern factors (EPFs) related to stomatal development. Prokaryotic expression vectors were constructed to obtain EPFs. Then the relationship between EPFs and hydrogen sulfide (H2S) was established. First, AtEPF1, AtEPF2 and AtEPFL9 were cloned and constructed to pET28a vectors. Then recombinant plasmids pET28a-AtEPF1, pET28a-AtEPF2 and pET28a-AtEPFL9 were digested and sequenced, showing successful construction. Finally, they were transformed into E. coli BL21(DE3) separately and induced to express by isopropyl β-D-galactoside (IPTG). The optimized expression conditions including IPTG concentration (0.5, 0.3 and 0.05 mmol/L), temperature (28 °C, 28 °C and 16 °C) and induction time (16 h, 16 h and 20 h) were obtained. The bands of purified proteins were about 18 kDa, 19 kDa and 14.5 kDa, respectively. In order to identify their function, the purified AtEPF2 and AtEPFL9 were presented to Arabidopsis thaliana seedlings. Interestingly, the H2S production rate decreased or increased compared with the control, showing significant differences. That is, EPFs affected the production of endogenous H2S in plants. These results provide a foundation for further study of the relationship between H2S and EPFs on stomatal development, but also a possible way to increase the yield or enhance the stress resistance.
Arabidopsis ; genetics ; metabolism ; Arabidopsis Proteins ; genetics ; isolation & purification ; metabolism ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Hydrogen Sulfide ; metabolism ; Plasmids ; genetics ; Seedlings ; metabolism

To construct a bait expression vector containing the duck circovirus Cap gene for use in the yeast two-hybrid system, the whole cap codon-optimized gene was inserted into pGBKT7 vector and confirmed by PCR, restriction enzyme digestion, and sequence analysis. After transformation into a Y2HGold yeast strain, the expression of Cap protein was analyzed by Western blotting. Toxicity and self-activation of the bait protein were detected using different dropout minimal base. PCR reaction, restriction enzyme digestion, and sequencing analyses indicated that the duck circovirus Cap gene was correctly inserted into pG- BKT7. Western blotting showed that the whole Cap protein was expressed. The recombinant bait protein had no toxicity and self-activation. Therefore, the bait vector with the Cap gene was constructed successfully, providing a foundation for future screening for interacting proteins in the yeast two-hybrid system.
Animals ; Capsid Proteins ; genetics ; metabolism ; Circovirus ; classification ; genetics ; isolation & purification ; Cloning, Molecular ; Ducks ; Genetic Vectors ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Two-Hybrid System Techniques

OBJECTIVETo clone, express, and purify cyclic diadenosine monophosphate (c-di-AMP) metabolism-related genes from Porphyromonas gingivalis (P. gingivalis) ATCC33277.

METHODSPolymerase chain reaction (PCR) from the genome of P. gingivalis ATCC33277 amplified, the coding regions of pgn0523, pgn1187, and pgn2003 genes. The amplified DNA fragments were ligated with a prokaryotic expression vector pET28a to construct the recombinant expression plasmids pET-pgn0523, pET-pgn1187, and pET-pgn2003. These recombinant plasmids were transformed into Escherichia coli (E. coli) BL21 (DE3) competent cells. The expression of recombinant proteins was induced by isopropyl-β-D-thiogalactoside and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were purified using a Ni²⁺ matrix column, and their concentrations were determined by a BCA Protein Quantitative Kit.

RESULTSThe c-di-AMP metabolism-related genes from P. gingivalis ATCC33277 were amplified successfully with the correct molecular size. The recombinant expression vectors were constructed by ligating enzyme-digested PCR products and pET28a vector, and verified by PCR and sequencing. After induction and purification, recombinant proteins were expressed successfully and obtained with the correct molecular size (19.5 x 10³, 39.9 x 10³, 66.0 x 10³). The final protein concentrations were 0.708, 0.523, and 0.861 mg · mL⁻¹ after dialysis.

CONCLUSIONThe c-di-AMP metabolism-related genes from P. gingivalis ATCC33277 are cloned successfully, and their coding products are expressed correctly in E. coli. High-purity proteins are finally obtained. The cloning and purification of these important proteins will help us to further investigate the physiological function and regulatory mechanism of c-di-AMP signaling system in P. gingivalis.

Bacterial Proteins ; biosynthesis ; genetics ; isolation & purification ; Cloning, Molecular ; Dinucleoside Phosphates ; Escherichia coli ; genetics ; Genetic Vectors ; Plasmids ; Polymerase Chain Reaction ; Porphyromonas gingivalis ; genetics ; Recombinant Proteins

OBJECTIVETo clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen.

METHODSVP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected.

RESULTSVP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay.

CONCLUSIONSVP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; Enterovirus A, Human ; genetics ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Hand, Foot and Mouth Disease ; immunology ; Humans ; Immunoglobulin M ; blood ; Recombinant Proteins ; genetics ; immunology

Fibroblast growth factor -21 (FGF-21) is a recently discovered metabolic regulation factor, regulating glucose and lipid metabolism and increasing insulin sensitivity. FGF-21 is expected to be a potential anti-diabetic drug. Expression of FGF-21 as inclusion bodies has advantages for high yield and purity, but the bioactivity of the protein is almost totally lost after denature and renature. That is why FGF-21 is currently expressed in soluble form. As a result, the yield is considerably low. In this study, we used SUMO vector to express SUMO-human FGF-21 (SUMO-hFGF-21) in form of inclusion body. We optimized the culture conditions to increase the yield of the bioactive human fibroblast growth factor-21. We applied the hollow fiber membrane filtration column to enrich the bacteria, wash, denature and renature inclusion bodies. After affinity and gel filtration chromatography, we examined the hypoglycemic activity of FGF-21 by the glucose uptake assay in HepG2 cells. We also detected the blood glucose concentration of type 2 diabetic db/db model mice after short or long-term treatment. The results show that the yield of ihFGF-21 was 4 times higher than that of shFGF-21. The yield was 20 mg/L for ihFGF-21 vs. 6 mg/L for shFGF-21. The purity of ihFGF-21 was above 95%, while shFGF-21 was 90%. Compared with the traditional method of extracting inclusion bodies, the production cycle was about three times shortened by application of hollow fiber membrane filtration column technology, but the bioactivity did not significantly differ. This method provides an efficient and cost-effective strategy to the pilot and industrial production of hFGF-21.
Animals ; Bacteria ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; Disease Models, Animal ; Fibroblast Growth Factors ; biosynthesis ; Genetic Vectors ; Glucose ; metabolism ; Hep G2 Cells ; Humans ; Hypoglycemic Agents ; isolation & purification ; Inclusion Bodies ; metabolism ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; Small Ubiquitin-Related Modifier Proteins ; biosynthesis

This paper is to report the development of a high-throughput in vitro system to screen hPXR/CAR mediated CYP2B6 drug inducers, and the application of it into the quick determination of induction activity toward CYP2B6 by various commonly used traditional Chinese medicines (TCMs) extract. Dual reporter gene assays were performed. The hPXR/CAR expression vectors and the reporter vector pGL3-CYP2B6-Luc involved in the distal and proximal promoters of CYP2B6 were co-transfected into HepG2 cells. Relative luciferase activities in cell lysate were analyzed after 48 h treatment of blank vehicle or drugs to determine the induction activity toward CYP2B6 by various commonly used TCMs extract. The positive hPXR/hCAR activators rifampicin and CITCO were applied to make sure that the reporter gene model was successfully established. Then 5 kinds of commonly used TCM extracts and 1 herbal compound were successfully investigated, some were found to activate hPXR or hCAR and therefore have the potential to induce CYP2B6 enzyme. This is the first domestic article to report the hCAR3-mediated CYP2B6 induction model and the establishment of a reporter gene system for hPXR/CAR-mediated CYP2B6 induction can be an effective and systemic in vitro method to investigate the drug inducers of CYP2B6 and to explain the mechanism involved.
Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Cytochrome P-450 CYP2B6 ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Genes, Reporter ; Genetic Vectors ; Hep G2 Cells ; High-Throughput Screening Assays ; Humans ; Luciferases ; genetics ; metabolism ; Oximes ; pharmacology ; Plants, Medicinal ; chemistry ; Plasmids ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Receptors, Steroid ; genetics ; metabolism ; Rifampin ; pharmacology ; Thiazoles ; pharmacology ; Transfection

The aim of this study is to establish stable transfected cell lines which could produce SPAG4L protein labeled with Myc and His tags in vitro. The open reading frame (ORF) of human SPAG4L was amplified by PCR and the fragments were cloned into eukaryotic expression vector pcDNA3.1/myc-His(-)A. The recombined plasmids of pcDNA3.1/myc-His(-)A/SPAG4L were verified by sequencing and digestion with enzymes. Then, the recombined plasmids were introduced into HeLa cells and screened by G418. Western blotting was performed to detect the expression of SPAG4L and its tags in stable transfected cell lines. SPAG4L and its tags were expressed in the stable cell lines transfected with pcDNA3.1/myc-His(-)A/SPAG4L, but not in the control group. Further study showed that SPAG4L colocalized with the endoplasmic reticulum(ER) marker PDI by immunofluorescence. The stable transfected cell lines established in this study will provide a powerful tool for further studies such as co-immunoprecipitation and pull-down.
Carrier Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Female ; Genetic Vectors ; genetics ; HeLa Cells ; Histidine ; genetics ; Humans ; Male ; Proto-Oncogene Proteins c-myc ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Transfection

The ORF sequence of glycosyltransferase gene BcUGT1 cloned from Bupleurum chinense DC. was analyzed and its three dimentional structure was predicted. Using qRT-PCR method, the expression characteristics of BcUGT1 after methyl jasmonate (MeJA) induction and in different plant tissues were investigated. The results showed that BcUGT1 may be involved in saikosaponin biosynthesis in B. chinense. Thereafter, the recombinant vectors of BcUGT1 were constructed for its expression in E. coli. The target protein was successfully expressed and purified. In the present study, three vectors, pRSET-A, pET-28a (+) and pET-30a (+), and three isolates of E. coli, BL21 (DE3) plysS, BL21A1 and BL21-CodonPlus (DE3)-RIPL were used under different induction conditions, such as different concentrations and during times of inducers (L-arabinose and IPTG) and different inducing temperatures. The results showed that in the condition of 0.5 or 1 mmol x L(-1) IPTG, 16 degrees C, 20 h, target protein expressed in BL21-CodonPlus (DE3)-RIPL with pET-28a (+) or pET-30a (+) as vector. Using PrepEase His-tagged protein purification kit, the target protein was purified. The present work will be helpful for follow-up bio-function analysis of BcUGT1.
Amino Acid Sequence ; Base Sequence ; Bupleurum ; chemistry ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Plant ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Glycosyltransferases ; genetics ; isolation & purification ; metabolism ; Oleanolic Acid ; analogs & derivatives ; biosynthesis ; Open Reading Frames ; genetics ; Phylogeny ; Plants, Medicinal ; chemistry ; Protein Structure, Secondary ; Recombinant Fusion Proteins ; genetics ; metabolism ; Saponins ; biosynthesis

The expression of phenylalnine ammonia lyase (LJPAL1) is closely related to the content of active compounds in Lonicera japonica. In this paper, a prokaryotic expression vector is constructed and LJPAL1 protein is expressed in E. coli. Three antigen sites were synthesized to peptide antigen and prepared polyclonal antibody of Anti-LJT-1, Anti-LJT-2 and Anti-LJT-3, separately. Antibody Anti-LJT-2 was screened using Western blotting. And indirect ELISA was built using Anti-LJT-2. The results of this study will be a base for honeysuckle chemical quality and evaluation kits.
Antibodies ; immunology ; metabolism ; Antibody Specificity ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Flowers ; chemistry ; Gene Expression Regulation, Plant ; Genetic Vectors ; Lonicera ; chemistry ; Phenylalanine Ammonia-Lyase ; chemistry ; immunology ; isolation & purification ; Plants, Medicinal ; chemistry ; Plasmids ; Protein Structure, Secondary ; Protein Structure, Tertiary