Objective： CD19 scFv (single-chain fragment variable) specifically recognizes CD19 molecule expressed on the B cells surface. Interleukin-10 (IL-10) -producing B cells are a major subset of regulatory B cells (Breg). The aim is to construct a recombinant fusion protein of CD19 scFv and IL-10 receptor1 (IL-10R1), which potentially captures and blocks IL-10 secreted by Breg cells.Methods: CD19scFv and IL-10R1 were cloned into pET-28a prokaryotic plasmid. Recombinant proteins were enriched and purified by Ni column, identified by SDS-PAGE and Western blot. Pull down analysis was performed to identify the proteins captured by CD19scFv-IL-10R1 recombinant protein.Results: The CD19 scFv-IL-10R1 fusion protein is prepared and the protein binds to both CD19 and IL-10 molecules in vitro.Conclusion: CD19scFv-IL-10R1 fusion protein binds to both CD19 molecule and IL-10 molecule in vitro, and has potential application as a specific inhibitor for Bregs.
The aim is proposed to understand the application of self-assembling peptide R2I4R2 3D cell culture human skin fibroblast in vitro and the role of the wound healing process. Circular dichroism was used to analysis its secondary structure influenced by different time,temperature and ion environment; Congo red staining was used to obtain the macro change of R2I4R2 hydrogel; R2I4R2 was used to 3D cell culture material to get the growth state of human skin fibroblast cell strain; AO / EB staining was used to study cell apoptosis in 3D culture environment;SD rat skin wound healing model was founded to check pathological changes by HE staining and immunohistochemical staining. These results indicated that R2I4R2 can form a stable secondary structure in some conditions;24 hours after self-assembling, R2I4R2 can form a homogeneous and stable membrane-like structure for 3D cell culture; Human skin fibroblasts had a hearty growing situation in hydrogel; The data in animal experiments show that R2I4R2 can reduce inflammation, promote neovascularization, accelerate the skin wound repair process. It demonstrate this short peptide can be used to a new material in 3D cell culture and skin trauma repairing and beyond.
The midgut is not only a digestive organ, but also a physiological barrier against invasive pathogens in silkworm, Bombyx mori. To clone and identify a novel midgut-specific promoter in silkworm, firstly detected the expression characteristics of a candidate gene BmP56 of tissue-specific expression by RT-PCR, and found that it midgut-specifically expressed. Furthermore, cloned its upstream regulatory region P56, and constructed a transgenic vector pBac[P56DsRedSV40,3×P3EGFP] of the expression of red fluorescence protein gene DsRed drived by the upstream regulatory region P56. By the way of microinjection and fluorescence screening, finally obtained the transgenic silkworm. The expression detection showed that the reporter gene DsRed only expressed in the midgut of transgenic silkworm, in consistent with the expression mode of BmP56, indicating that the upstream regulatory region P56 is an active, midgut-specifcally expressed promoter in silkworm.
Cinnamoyl-CoA reductaseis the first rate limiting emzyme which catalyzes the biosynthesis of lignin and plays an important role in lignin biosynthesis. Two cinnamoyl-CoA reductase encoding genes, CiCCR2 and CiCCR3, were cloned from Caragana intermedia through PCR. The open reading frame(ORF) of CiCCR2 is 897bp which encoding 299 amino acids, and ORF of CiCCR3 is 966bp, which encoding 322 amino acids. Quantification assay and Histochemical staining showed that the lignin content increased by overexpressing of CiCCR2 and CiCCR3 in Arabidopsis compared with the wild type during both seedling and mature stages. At the same time, the fresh and dry weight of overexpressing plants were higher than that of the wild type.
R-mandelate dehydrogenase plays a key role in the biosynthesis of phenylglyoxylic acid, thus exploiting nove R-mandelate dehydrogenase with higher catalytic activity and stability has significant economic value. In order to obtain the perfect R-mandelate dehydrogenase, a novel R-mandelate dehydrogenase was obtained by genome mining, named as LhDMDH, which was from Lactobacillus harbinensis. The specific activity of LhDMDH was 1264.3 U/mg, which was near to four times that of the probe and leading in the reported enzyme. Meanwhile, the main enzymatic characterizations of the four recombinant enzymes were researched. Their temperature optima were 25 to 30 ℃, and their pH optima were 9.0 to 9.5. The Kcat of LhDMDH is 30.28 S -1, which is obviously higher than the others. In addtion, the results of substrate spectrum of R-mandelate dehydrogenases indicated that the LhDMDH could have advantages over other enzymes in chiral resolution of racemic mandelic acid and the biosynthesis of phenylglyoxylic acid. This gained ideal results in genome mining of R-mandelate dehydrogenases, established a solid foundation for further transformation and application, and provided a useful experience for the exploiting of other enzymes.
Candida tropicalis1798 can produce glycerol when ferment grease. C.tropicalis1798 efficient use of glycerol and it will provides energy for fermentation and improve the utilization of grease by genetically modified. A related glycerol metabolism gene gk in C. tropicalis 1798 was intended to replace the promoter gene about 500bp DNA fragment gkpR in gk gene was cloned by using PCR, gkpR and a G418 resistance gene (kan r) was connected to vector of pPICzαA by One Step Cloning Kit,the promoter gene of pGAP from C. lipolytica 1457 will be connected to the gkpR in recombinant vector by One Step Cloning Kit. The recombinant plasmid pPICzαA-gkp was transformed into C. tropicalis 1798 competent cells.Through a single homologous exchange, the promoter pGK was replaced by pGAP. After G418 resistance experiments, PCR determination, the pGAP promoter replacement C. tropicalis 1798-gkPr was obtained successfully;The verification results offer mentation shows, The analysis founded that the OD600 of C. tropicalis 1798-gkPr was 46.4% higher than that of C. tropicalis 1798 and the glycerol content of C. tropicalis 1798-gkPr was accounted for just 56.1% when it was cultured for 12 hours with glycerin as substrate. It revealed that the replacement of promoter pGAP gene affected utilization of glycerol in C. tropicalis 1798-gkPr. Another analysis also founded that the long-chain dicarboxylic acid production of C. tropicalis 1798-gkPr was 32.7% higher than that of C. tropicalis 1798 when it was cultured for 6 days with oil as substrate. Conclusion: A C. tropicalis 1798 strain with the replaced promote gene was successfully constructed and it increased the utilization of C. tropicalis 1798 for glycerol constituents in the grease component.
Polyhydroxyalkanoates (PHAs) have received considerable attention because of their material properties and wide applications for packaging, biofuels, and biomedicine.Polylactate (PLA) isarepresentative bio-based polyester, whichis chemically synthesized rather than synthesized bybacteria, because of the substrate specificity of PHA synthase (PhaC). To produce lactate-based polyesters, itiscriticalto obtain a PhaC capable of LA-polymerization. Glycerol dehydratase DhaB123 and its reactivating factorGdrAB from Klebsiella pneumoniae, and propionaldehyde dehydrogenase PduP from Salmonella typhimurium LT2 were overexpressed to convert glycerol into 3-hydroxypropionyl-CoA;propionyl-CoA transferase (PCT) from Megasphaera elsdenii DSM 20460were used to obtain lactyl-CoA; and three different PhaC were introduced and examined for poly (3-hydroxypropionate-co-lactate) (P(3HP-co-LA)). The wild type enzyme PhaC1, PhaC2 from Pseudomonas putida can’t polymerize lactyl-CoA into polymer. Only with double mutant (Ser325Thr and Gln481Lys) PhaC1(STQK), 3HP-CoA and lactyl-CoA were copolymerized into P(3HP-co-LA)in engineered Escherichia coli. Under optimal conditions, theP(3HP-co-LA) production and contentwere 0.22 g/Land 3.2%(wt/wt[celldryweight]), respectively.A good example was provided for lactate-basedbiopolymerby biosynthesis.
Objective： Exploration of the purification efficiency of proteins in prokaryotic expression system(Escherichia coli) by using an elastin-like protein tag(ELP30-tag) with small molecular weight.Methods: The ELP30-tag gene was synthesized and inserted into the pET-28a(+) vector,two intein genes (intein1 & intein2) and an enhanced green fluorescent protein (eGFP) gene were cloned and applied to construct four prokaryotic expression vectors: pET-ELP30, pET-ELP30-eGFP, pET-ELP30-intein1-eGFP and pET-eGFP-intein2-ELP30. All the recombinant plasmids were transferred into E. coli BL21(DE3)and induced by IPTG, respectively. Recombinant proteins ELP30, ELP30-eGFP,ELP30-intein1-eGFP and eGFP-intein2-ELP30 were purified by inverse transition cycling (ITC), and then the cleavage reaction of intein1 and intein2 were induced by adjusting the pH value of the solution or adding DL-Dithiothreitol (DTT), respectively, and last the pure eGFPs were separated by one more ITC reaction.Results: The recombinant proteins ELP30, ELP30-eGFP and eGFP-intein2-ELP30 were purified by using the designed ELP30-tag; the cleavage reaction of inteins from the recombinant proteins ELP30-intein1-eGFP and eGFP-intein2-ELP30, which could be successfully induced,and then the eGFPs were released into the solution but not separated. This lays some foundations for the application and optimization of the ELP-tags with small molecular weight.
Objective: Using c-Myc-GST protein as target molecule, nanobodies that can specifically recognize c-Myc tags (EQKLISEEDL) were screened from phage displayed immune libraries.Methods: Screening of phages specific bingding c-Myc-tag by solid-phase biopanning technology. Positive clones were identified by phage ELISA and then sequenced. The DNA fragment that coded positive nanobody phages were subclone to pET25b(+)vector ,the recombinant expression vector transformed into E.coli Rosetta (DE3) cells for expression under induction of IPTG. SDS-PAGE was used to analysis of recombinant protein expression. Finally, the binding activity and specificity of the nanobodies were confirmed by indirect ELISA and quantum dot immunofluorescence technology. Result: After four cycles solid-phase biopanning, phage with binding activity clones were effectively enriched, the recovery rate was improved by 145 times and positive rate increased from 20.83% to 85.4%. The nanobodies A25 and A26 with high OD450 in phage-ELISA were recombinantly expressed , the production yield was 60 mg/L. Indirect ELISA results indicate that the recombinant proteins A25 and A26 can recognize the c-Myc-tag. Quantum dot immunofluorescence technology results showed that A25 could detect c-Myc protein in SP2/0 cells.Conclusion: Anti-c-Myc tagged nanobodies were successfully screened and two prokaryotic expression vector were constructed, the recombinant proteins achieved souble expression. And these laid the foundation for the detection of intracellular c-Myc protein.
2,3-butanediol is used widely. As a potential platform compound, it can be used to replace the traditional platforms—four carbon hydrocarbons. With the requirements of energy security and environmental protection, biological refining 2,3-butanediol gets the substantial interest of researchers, which has obvious advantages over chemical method. Therefore, it is important to develop the suitable fermentation process of 2,3-butanediol. With the strain Klebsiella pneumoniae CICC10011, a preliminary study on the properties of 2,3-butanediol fermentation was carried out. By changing conditions, researchers studied the effects of pH, air flow rate and rotating speed on the metabolism of the stain. In this way, the better fermentation conditions for producing 2,3-butanediol were got. In the process, pH, air flow rate and rotating speed were controlled through two-stages. In the first 12h, kept the pH 6.8,the air flow rate 1.0vvm and the rotating speed 400r/min. Then, changed them to 6.0, 0.5vvm and 300r/min, respectively.
Peptide nucleic acid (PNA) is a synthetic analogue of DNA with the pseudopeptide backbone, which has the advantages of strong binding specificity with nucleic acid, high biological stability in tissues and cells and long half-life.By targeting DNA/RNA binding, PNA could inhibit the replication, transcription and translation of DNA/RNA.Chiral functional groups were introduced at gamma sites of the skeleton structure of PNA to form right-handed helical structures, which significantly improved the hybridization with targeting DNA/RNA, and this PNA derivative was called gamma PNA.The solubility, thermal stability and specificity of gamma PNA have been improved remarkably, and it has a good prospect in gene editing and probe detection. The structure, properties and research progress of gamma PNA are summarized, which provides theoretical basis and references for antisense PNA application.
Circular RNAs (circRNAs) are a newly discovered RNA molecules, which are widely distributed in mammalian cells. They do not have 5'cap and 3' tail, but have a high degree of sequence conservation and stability, and not easy to be degraded by RNA exonuclease. What’s more, they not only can regulate the expression of genes, but also play a regulatory role at transcriptional or transcriptional levels. In recent years, circRNAs have been found to be associated with many tumors including gastric cancer. The formation, classification, molecular characterization and function of circRNAs were introduced. Then, the relationships between circRNAs and gastric cancer are analyzed from three aspects: the correlations between circRNAs and gastric cancer occurrence, the values of circRNAs in the diagnosis of gastric cancer, and the roles of circRNAs in the treatment of gastric cancer.
Antibodies trigger unwanted immune response, which influence the efficacy and safety of drugs. Therefore, it is necessary to evaluate immunogenicity of antibodies in different stage of development, including post-marketing surveillance. Immunogenicity evaluation is the key step in the development of all antibodies. The regulatory authorities have strict requirements on the immunogenicity of antibodies products for evaluation, there is a lack of uniform standards for the type, quantity, and quality of evidence,but also the lack of immunogenicity of antibodies guidance documents or immunogenic experimental design standards. New technologies promote the development of immunogenicity evaluation, the detection rate of immunogenicity also rise,therefore “head to head” clinical trials to evaluate the immunogenicity of antibodies. Therefore, the regulators and clinicians need to be aware of the change of immunogenicity analysis. Here, the related factors of immunogenicity of antibodies, potential clinical consequences, changes of the current regulatory guidance and development of non clinical and clinical studies of immunogenicity evaluation methods were discussed. And a special note of immunogenicity evaluation on biosimilars was also discussed.
Mycoplasmal pneumoniae of swine (MPS) is a chronic respiratory infectious disease caused by Mycoplasma hyopneumonia (Mhp), thus causes huge losses to the pig industry. At present, vaccination is the main means to reduce or prevent the economic losses caused by Mycoplasma pneumoniae. The research progress of gene engineering vaccine of Mycoplasmal pneumoniae of swine in recent years is discussed, mainly includes adhesion factors related vaccines, ribonucleotide reductase related vaccines, DNA vaccines, expression library vaccines and peptide vaccines, etc. Finally, suggestions for the development of genetic engineering vaccines are proposed.
3’,5’-cyclic adenosine monophosphate (cAMP), an important and active compound due to its participation in various physiological actions, acts as a key second messenger. It was first reported in 1957. Since then a lot of research had been carried on for its numerous functions. Exogenous cAMP was developed as pharmaceuticals early in 1970s and it has also been proved to have a great potential in animal husbandry. Now the known cAMP API (Active Pharmaceutical Ingredient) was all produced by chemical synthesis. On the other hand, the species used by microbial cAMP fermentation research includes Arthrobacter, Bacillus subtilis and yeast. As a result of utilizing metabolic regulation mechanism and technology, the extracellular cAMP level could reach ≥7.23 g/L for Arthrobacter and 6~7 g/L for Bacillus subtilis. High levels of extracellular cAMP production by Saccharomyces cerevisiae were reported in recent years. In order to make full use of microbial fermentation and realize its industrial production, it needs to further improve the performance of fermenting microorganisms and the fermentation technology, solve the scale-up problem and make the process more economic.
Technologies for the production of biothanol are receiving increased attention around the world owing to concerns over the global warming. Lignocellulosic biomass is a great potential resource for the production of biofuels because it is largely abundant, inexpensive and renewable organic material. Significant efforts, many of which have been successful, have been made to convert these lignocellulosic biomass to valuable products, such as biofuels. Sustainable development in lignocellulosic bioethanol production has major challenge due to high cost of production. There are several issues such as efficient utilization of pentose sugars present in lignocelluloses, economical production of lignocellulolytic enzymes with high specificity, cost-effective pre-treatment of lignocellulosic biomass, etc. Genetically modified yeast strains have been approached to utilize pentose and hexose sugars for bioethanol production. However, these strains showed limited xylose consumption. Saccharomyces cerevisiae rely on the capacity of endogenous hexose transporters for xylose uptake, since S. cerevisiae lacks a xylose-specific transport system. Hence, there are several strategies that have been applied to engineer the yeasts which could improve the xylose transportation. latest advancements in S. cerevisiae xylose transporter genes were discussed.
As the most widely planted crops, genetically modified soybean plays an important role in the guarantee of human oil and feed supply. By using the method of patent analysis, a statistical research on the patent documents from PatSnap database in the field of genetically modified soybean in 1985~2016 was accomplished, the results showed the overall development trend of the global soybean patents, the research hot spot and the technology distribution and pattern. A comparative analysis of the competitiveness of China’s soybean research and development was also carried out. The future of soybean industry development was finally put forward.
