Advances in meningococcal polysaccharide conjugate vaccine research / 中国生物制品学杂志

@#Meningococcal polysaccharide conjugate vaccine is basically produced by chemical combination,and the most commonly used method is amide condensation reaction.Because of the covalent bonds between polysaccharide and protein,the prepared conjugate vaccine has high stability and good technical advantages,which plays an important role in the prevention of meningococcal related diseases.The vaccine can be applied to the immunization of young children under 2years old,and has more lasting protective effect.In this paper,the factors influencing the preparation of meningococcal polysaccharide conjugate vaccine,the binding mode of polysaccharide and carrier protein,the present situation at home and abroad and the existing problems in the preparation process were reviewed.

Advances in meningococcal polysaccharide conjugate vaccine research / 中国生物制品学杂志

@#Meningococcal polysaccharide conjugate vaccine is basically produced by chemical combination,and the most commonly used method is amide condensation reaction.Because of the covalent bonds between polysaccharide and protein,the prepared conjugate vaccine has high stability and good technical advantages,which plays an important role in the prevention of meningococcal related diseases.The vaccine can be applied to the immunization of young children under 2years old,and has more lasting protective effect.In this paper,the factors influencing the preparation of meningococcal polysaccharide conjugate vaccine,the binding mode of polysaccharide and carrier protein,the present situation at home and abroad and the existing problems in the preparation process were reviewed.

Target-specific gene delivery in plant systems and their expression: Insights into recent developments

High-yielding Indian cotton varieties are not amenable for regeneration and transformation because they arerecalcitrant in nature. In this work, we have developed Narasimha (NA1325) cotton variety by introducing threeCrygenes driven by three different promoters conferring insect resistance. The meristematic region of embryo axisexplants were infected and co-cultivated with Agrobacterium tumefacience (LBA4404) harbouring pMDC100vector with three Cry gene cassettes (a-globulin : Cry2Ab, DECaMV35s : Cry1F and nodulin : Cry1Ac) with Npt IIas a selectable marker gene. Out of 1010 embryo axes explants infected, 121 (T0) regenerated under two rounds ofkanamycin selection medium. About 2551 T1 seeds were collected from 111 T0 plants and these seeds screened againwith kanamycin at seedling stage. The transgenic plants were characterized by PCR, real time quantitative PCR,lateral flow strip protein assay and insect bioassay. Out of 145 kanamycin resistant plants (T1), twelve showedamplification of all four transgenes: Npt II, Cry2Ab, Cry1F and Cry1Ac through PCR with expected amplicons as395, 870, 840 and 618 bp, respectively. Further, lateral flow strip test revealed Cry1F and Cry1Ac proteinsaccumulated in 12 plants, whereas Cry2Ab protein was detected in eight only. The transcripts of all three Cry geneswere accumulated significantly higher in transgenic plants at T2 generation. The transgenic lines showed effectiveresistance against Helicoverpa armigera and Spodoptera litura larvae. The T2 line L-3 exhibited highest percentageof insect mortality, in which transcripts of all cry genes were accumulated higher than other plants. The transgeniccotton plants carrying triple Cry genes could be an excellent germplasm resource for the breeders for introgressions.

Identification and functional analysis of soybean stearoyl-ACP Δ⁹ desaturase (GmSAD) gene family / 生物工程学报

Stearoyl-ACP Δ⁹ desaturase (SAD) catalyzes the synthesis of monounsaturated oleic acid or palmitoleic acid in plastids. SAD is the key enzyme to control the ratio of saturated fatty acids to unsaturated fatty acids in plant cells. In order to analyze the regulation mechanism of soybean oleic acid synthesis, soybean (Glycine max) GmSAD family members were genome-wide identified, and their conserved functional domains and physicochemical properties were also analyzed by bioinformatics tools. The spatiotemporal expression profile of each member of GmSADs was detected by qRT-PCR. The expression vectors of GmSAD5 were constructed. The enzyme activity and biological function of GmSAD5 were examined by Agrobacterium-mediated transient expression in Nicotiana tabacum leaves and genetic transformation of oleic acid-deficient yeast (Saccharomyces cerevisiae) mutant BY4389. Results show that the soybean genome contains five GmSAD family members, all encoding an enzyme protein with diiron center and two conservative histidine enrichment motifs (EENRHG and DEKRHE) specific to SAD enzymes. The active enzyme protein was predicted as a homodimer. Phylogenetic analysis indicated that five GmSADs were divided into two subgroups, which were closely related to AtSSI2 and AtSAD6, respectively. The expression profiles of GmSAD members were significantly different in soybean roots, stems, leaves, flowers, and seeds at different developmental stages. Among them, GmSAD5 expressed highly in the middle and late stages of developmental seeds, which coincided with the oil accumulation period. Transient expression of GmSAD5 in tobacco leaves increased the oleic acid and total oil content in leaf tissue by 5.56% and 2.73%, respectively, while stearic acid content was reduced by 2.46%. Functional complementation assay in defective yeast strain BY4389 demonstrated that overexpression of GmSAD5 was able to restore the synthesis of monounsaturated oleic acid, resulting in high oil accumulation. Taken together, soybean GmSAD5 has strong selectivity to stearic acid substrates and can efficiently catalyze the biosynthesis of monounsaturated oleic acid. It lays the foundation for the study of soybean seed oleic acid and total oil accumulation mechanism, providing an excellent target for genetic improvement of oil quality in soybean.

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB-AcpM

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB-AcpM

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

Increasing pinosylvin production in Escherichia coli by reducing the expression level of the gene fabI-encoded enoyl-acyl carrier protein reductase

Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)- VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.

The differential diagnostic value of pleural effusion heparin-binding protein in parapneumonic effusion / 中华检验医学杂志

Objective The aim of this study was to evaluate the value of pleural effusion heparin-binding protein ( HBP) in differential diagnosis of parapneumonic effusion .Methods Case-control study. The pleural effusion of 189 patients with pleural effusion admitted to Quzhou People's Hospital from February to July 2018, including parapneumonic effusion (n=72), tuberculous pleural effusion (n=24), cases of malignant pleural effusion ( n=46 ) and transudative pleural effusion ( n=47 ) were collected.Routine analysis,lactate dehydrogenase(LDH),adenosine deaminase (ADA) and total protein(TP)examination of all pleural effusions were performed .The levels of heparin-binding protein in the patients'pleural fluid were measured by ELISA.The difference in the overall level of each group was determined by One-way ANOVA or LSD test followed by Kruskal-Wallis H test dependence on the homogeneity of variances .The categorical data was analyzed by chi-square test.Receiver operating characteristic ( ROC) curve was plotted to evaluate the diagnostic value of heparin-binding protein for parapneumonic effusion . Results The concentration of heparin-binding protein was low in malignant pleural effusion [15.2(8.4, 33.3) ng/ml] and transudative effusion[14.1(6.5, 23.0)ng/ml], but high in parapneumonic effusion[316.1(99.5,399.8)ng/ml]and tuberculous pleurisy [64.7 (18.6, 96.8) ng/ml] .The heparin-binding protein level in parapneumonic effusion was significantly different from the other three groups (H=120.3,P<0.05).The receiver operating characteristic curve analysis for an optimal discrimination between parapneumonic effusion from non -parapneumonic effusion could be performed at a cut-off point of 64.2 ng/ml with area under the curve of 0.953[sensitivity:88.9％(64/72), specificity:89.7％(105/117),positive predictive value:84.2％(64/76), negative predictive value:92.9％(105/113)].Conclusions Heparin-binding proteinin pleural fluid is effective to be used to classify parapneumonic effusion samples .The detection of heparin-binding protein in pleural effusion has good sensitivity and specificity .It could be a biomarker for differential diagnosis of parapneumonic effusion .

The research progress of uric acid metabolism in intestine / 医学研究生学报

Urid acid is one of the terminal metabolites of human body.More and more attention was paid to its metabolic mechanisms in intestinal tract;however, few studies were seen so far.This study aims to illuminate the metabolic mechanism of uric acid in intestinal tract by two ways:one is the transporters associated with intestinal epithelium, including ABCG2 and SLC2A9, the other is decomposition of uric acid by intestinal flora.We hope this review can provide new insights to decrease blood uric acid and treat urate-related diseases, and also provide a new way to alleviate drug-induced kidney damage.

Relationship between reduced folate carrier gene methylation and methotrexate resistance / 国际儿科学杂志

Tumor recurrence and drug resistance impact patients'therapeutic effect and prognosis seriously.In recent years,it has been found that epigenetic changes are involved in drug resistance.DNA methylation is one of the ways of epigenetic.DNA methylation of drug metabolism enzyme gene can affect gene's expression,leading to drug resistance.Methotrexate(MTX),a folic acid antagonist,is the most widely used drug in the treatment of malignant tumors,such as leukemia,osteosarcoma,head and neck cancer,and so on.Reduced folate carrier(RFC) is the main transporter cartier of MTX,and gene methylation is related to tumor cells'resistance to MTX.This paper reviews the role of RFC,relation of RFC methylation and methotrexate resistance and the factors of RFC methylation.

Preparation and identification of norepinephrine complete antigen and study on its immunogenicity in mice / 中华微生物学和免疫学杂志

Objective To construct and identify norepinephrine ( NE) complete antigen for the preparation of high sensitive and specific anti-NE monoclonal antibody .Methods Glutaraldehyde ( GA) and 1-Ethyl-3-(3-Dimethylaminopropyl ) carbodiimide ( EDC) were used to cross-link NE with carrier pro-teins (BSA, OVA) for NE complete antigen preparation under conditions of pH 4.5 or pH9.0.Three assays including UV scanning , SDS-PAGE and FeCl3 color reaction were performed for identification of NE com-plete antigen.Serum antibody titers were evaluated in mice model induced by intraperitoneal immunization with NE complete antigen .Results NE complete antigens were successfully prepared as indicated by the three identification assays .The coupling ratio was significantly increased in a time-depended manner under the condition of pH9.0 in comparison to that in the condition of pH 4.5.Indirect ELISA results showed that , when coating antigens and serum antibodies were prepared with the same cross -linking method , the serum antibody titers were significantly higher than those with different methods .Conclusion Anti-NE antibodies were successfully prepared by immunizing mice with NE complete antigens .

Docking studies on novel alkaloid tryptanthrin and its analogues against enoyl-acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis.

Isoniazid resistance is a serious threat in the battle against the treatment of multi-drug resistant tuberculosis (MDR-TB) and extremely drug-resistant tuberculosis (XDR-TB). Isoniazid is an inhibitor of enoyl-acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis, which is an important and functional enzyme of the type II fatty acid synthesis system and important therapeutic target. Natural alkaloid tryptanthrin and its analogues have shown anti-tubercular activity against MDR-TB, but their cellular target is unknown. In this work, in silico molecular docking was performed using docking server in order to see the interaction of tryptanthrin and its 15 analogues with InhA of M. tuberculosis. Results showed that among tryptanthrin and its 15 analogues, tryptanthrin and its two analogues exhibited good affinity to the binding site of InhA with free binding energy of -7.94 kcal/mol and inhibition constant (Ki) of 1.50 µm. Active site residues of InhA interacting with tryptanthrin were Ser13, Thr39, Phe41, Leu63, Asp64, Val65, Ile95, Phe97 and Ile122. In binding mode, polar bond were found between O1 (1) with Asp64 of bond length (3.34 Å) and hydrophobic bonds were found between Leu63 with C15 and C12, Val65 with C7, Val65 with C12 and C4, Ile95 with C6 and C7, Ile95 with C10, C12 and C14. Important pi-pi bonds were found between Phe41 with C2, C5, C7, C12, C4, C6, C8, C9, C13 and Phe97 with C9. These interactions indicated stability of tryptanthrin in active residue and suggested it as a potential drug candidate. Thus, good affinity of tryptanthrin to binding site of InhA may lead to synthesis of anti-tubercular drug capable of combating MDR strains of M. tuberculosis

Purification and characterization of riboflavin carrier protein from egg white of South Indian spotted owlet (athene brama).

Riboflavin carrier protein has been isolated and purified from the Indian spotted owlet. The protein was purified to its homogeneity. Purification was achieved successfully by DEAE_ Sepharose column chromatography and gel filtration chromatography on Sephadex G-100. The protein content was estimated with Lowry method. The purity of the proteins was judged by SDS-PAGE technique. The molecular weight of the protein was found to be 29 Kd. The protein was characterized using absorption, fluorescence and CD spectral analysis. Significance of the above results are discussed in the present communication.

Characterization of ?-Ketoacyl-acyl Carrier Protein Synthase Ⅱ Homologues in Enterococcus faecalis / 生物化学与生物物理进展

FabB(?-ketoacyl-acyl carrier protein synthase Ⅰ)and FabF(?-ketoacyl-acyl carrier protein synthase Ⅱ)are two key enzymes of fatty acid biosynthesis in E.coli.The Gram-positive pathogenic bacterium Enterococcus faecalis has a fatty acid composition very similar to that of E.coli.Bioinformatic analysis reveals that though E.faecalis has two fabF homologues,there is no recognizable fabB homologue in the genome of E.faecalis.Two fabF homologues(fabF1 and fabF2)were amplified by using E.faecalis V583 genomitic DNA as template,and two plasmids,pHW13(fabF1)and pHW14(fabF2),were constructed.The results of experiments in vivo and in vitro have shown that fabF1 gene could complement E.coli fabB mutation and FabF1 possessed ?-ketoacyl-acyl carrier protein synthase Ⅰ(FabB)activity,while fabF2 gene could complement E.coli fabF mutation and FabF2 had ?-ketoacyl-acyl carrier protein synthase Ⅱ(FabF)activity.Meanwhile the data also shown that FabF2 possessed partial function of ?-ketoacyl-acyl carrier protein synthase Ⅰ(FabB),and it could make E.coli fabB mutation synthesized low amount of unsaturated fatty acid.From these data it is clear that FabF species enzymes could have activity of ?-ketoacyl-acyl carrier protein synthase Ⅰ(FabB).

Inhibitory Effect of Piperine on Gallstone Formation in the Gallbladder of C57BL/6 Mice / 中国中医药信息杂志

Objective To observe the effect of piperine (PA) on experimental gallstone formation in the gallbladder of C57BL/6 mice. Methods 3 dietary groups of C57BL/6 with 10 mice each group were allocated as control (normal mice chow),lithogenic (1% cholesterol diet) and PA (1% cholesterol diet + PA 30 mg/kg body weight) group respectively for 4 weeks. The expression of Scp2 gene in liver tissue was measured by RT-PCR and the bile lipid contents was measured chemically,the cholesterol saturation index (CSI) was calculated by Carey’s method. Results Cholesterol crystals and stones were found in 10/10 and 9/10 gallbladders respectively in lithogenic group,but not found in control and PA groups. Comparing with the lithogenic group,the expression of Scp2 gene in liver tissue and CSI in the gallbladder bile were significantly decreased in PA group. Conclusions PA inhibit the experimental gallstone formation induced by high cholesterol feeding in C57BL/6 mice,with simultaneous decreasing of both the Scp2 gene expression in the liver tissue and the CSI value in the gallbladder bile. The further study of the preventive effect of gallstong formation of PA,whether or not related to the above results,is strongly suggested.

Synthesis and Identification of the Antigens for Ciprofloxacin / 中国兽医学报

Hapten-carrier protein conjugates were made using ciprofloxacin (CPFX) and two carrier proteins by 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) method. Ultraviolet spectrophotometry were used to demonstrated that the molecule conjugate ratio of CPFX to ovalbumin (OVA) and bovine serum albumin (BSA) are 6:1 and 13:1 respectively. Nondenaturing gel electrophoresis results revealed that the conjugate band migrates differently from that of the carrier protein alone and of the EDC-treated protein when as few as 6 molecules of CPFX are attached to the carrier protein. The results indicate that nondenaturing gel electrophoresis and ultraviolet spectrophotometry can be employed to analyze the molecule coupling ratio of CPFX to carrier proteins qualitatively and quantitatively.

Comparison of 2 kinds of construction and expression methods of reconstructed human cathelicidin LL-37 / 第三军医大学学报

Objective To employ 2 approaches to construct and express reconstructed LL-37 (rLL-37) in procaryotic system, and to explore a better preparation method. Methods The first method: the rLL-37 was inserted into vector pET-28a (+), then was induced to express in E.coli. BL21 (DE3) and purified by chromatography; the second method: the rare codons in the rLL-37 gene sequence were substituted by the preferred codons of procaryotic cell, and a fragment of carrier protein molecule (CPM) was added to the N termination of the objective sequence to construct expression plasmid pET-30a(+)-CPM-rLL-37, then the rLL-37 was expressed in E.coli. BL21 Star(DE3) and purified by chromatography. The productive rates of the 2 methods were compared and the antimicrobial effects of obtained rLL-37 was studied. Results The first method: the DNA sequence of rLL-37 was obtained successively by Touch-Down PCR. The expression plasmid pET-30a(+)-CPM-rLL-37 was expressed with fusion protein in E.coli BL21 (DE3). The expression rate accounted for 20% of total bacterio-protein, then the expressed product was purified by using high positive ion exchange column Macro-Prep High S; The second method: a fragment of carrier protein molecule was designed that contained 28 amino-acid residue and its pHi was 2.7, net charge was-6.0 at pH 7.4. After the expression plasmid pET-30a(+)-CPM-rLL-37 was constructed successively, it was expressed in E.coli BL21 Star (DE3). The expressed fusion protein accounted for 35% of total bacterio-protein, then the expressed product was purified by using affinity binding chromatography with TALON resins successfully. The obtained 2 kinds of rLL-37 were able to kill both Gram-negative and-positive bacteria by the means of inhibitory zone. Conclusion It’s feasible to prepare efficiently rLL-37 in procaryotic system, which founds the basis for the further research on bactericidal activity of rLL-37.

Synthesis of huperzine A-bovine serum albumin conjugate and its immunogenicity in mice / 第三军医大学学报

Objective To prepare huperzine A-carrier protein conjugate and determine its immunogenicity for later production of monoclonal antibody against HupA and establishing an enzyme-linked immunosorbent assay ( ELISA) -based detection method for HupA. Methods HupA was conjugated with bovine serum albumin ( BSA) using glutaraldehyde ( GA) method to obtain the HupA-GA-BSA conjugate,and the hapten number in the conjugate was determined by matrix-assisted laser absorption ionization time-of-flight mass spectrometry ( MALDI-TOF-MS) . BALB /c mice were immunized with HupA-GA-BSA to prepare the antiserum against HupA. The serum titer and specificity of the HupA antibodies were detected by indirect ELISA and competitive ELISA,respectively. Results The conjugation ratio of HupA and the carrier protein BSA was 8∶ 1. The antiserum against HupA with a titer of 1∶ 62 500 was obtained after immunizing the mice with the conjugate,and the antiserum reacted specifically to HupA. Conclusion The synthesized HupA-GA-BSA conjugate possesses good immunogenicity in mice,suggesting the feasibility of preparing the monoclonal antibody against HupA using this conjugate.

Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies.

Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.

Cloning and Expression of Acyl Carrier Protein Gene from Schizochytrium / 微生物学通报

Acyl carrier protein is an essential component involved in the biosynthesis of DHA(Docosahexaenoic Acid) via PKS(Polyketide synthase) pathway,which takes the growing acyl chain from one enzyme to another.One cDNA clone,with high homology of ACP,was isolated from Schizochytrium sp.FJU-512 cDNA library.The deduced amino acid sequence contained 142 residues with isoelectric point of 5.04 and had the 4'-phosphopantetheine prosthetic(4'-PP) binding site.The target fragment was digested with BamHⅠ/HindⅢand inserted into the expression vector pET-30a resulting in the plasmid pET-30a/acp.The recombinant vector was transformed into E.coli BL21(DE3) and induced by IPTG.SDS-PAGE analysis demonstrated that ACP was effectively expressed.