Objective: To investigate the role and mechanism of chemokine receptor CX3CR1 in the regulation of osteogenic differentiation of human aortic valve interstitial cells, and to provide new ideas for early intervention and treatment of calcific aortic valve disease.Methods: Three non-calcified aortic valves and five calcified aortic valveswere taken.Immunohistochemical staining was used to detected the expression of osteogenic-related transcription factors Runx2,osteopontin (OPN) andosteocalcin (OCN).Three non-calcified aortic valves were taken to culture human aortic valve interstitial cells were isolated by collagenase digestion.Cell morphology and growth status were observed,and phenotypic identification was performed by immunofluorescence staining.The overexpression plasmid CX3CR1 were added into the osteoblast-induced culture of human aortic valve interstitial cells (CX3CR1+OM),the normal complete medium culture (CM) group,the osteogenic induction medium culture (OM) group, and the negative control plasmid was added into the osteogenic induction medium (negative control+OM) group were arranged in parallel.the expression of Runx2,OPN and OCN was detected by qPCR and Western blot, the expression of AKT and p-AKT was detected by Western blot.Alizarin red S staining was used to evaluate the formation of advanced calcium nodules.The interference plasmid siCX3CR1 were added into the osteoblast-induced culture of human aortic valve interstitial cells(siCX3CR1+OM), the normal complete medium culture (CM) group, the osteogenic induction medium culture (OM) group, andthe negative control siRNA was added into the osteogenic induction medium (negative siRNA control+OM) group were arranged in parallel. The expression of Runx2, OPN and OCNwere detected by qPCR and Western blot,the expression of AKT and p-AKT were detected by Western blot.Alizarin red S staining was used to evaluate the formation of advanced calcium nodules.Results: Clinical specimens showed that calcified valves had higher expression of CX3CR1 than non-calcified valves (P<0.05). Successful isolation of human aortic valve interstitial cells, stromal cell-specific marker protein smooth muscle actin α-SMA and Vimentin positive, endothelial cell specific marker protein vWF is negative. Compared with the CM, OM,and negative control group, CX3CR1+OM group have the expression of Runx2, OPN and p-AKT (P<0.05), and alizarin red S staining shows obvious calcium nodules. Compared with the CM,OM,negative siRNA control+OM group, siCX3CR1+OM shows a down-regulated expression of Runx2,OPN and p-AKT (P<0.05), Alizarin red S staining shows a decrease in calcium nodules.Conclusion: The chemokine receptor CX3CR1 promotes osteogenic differentiation of human aortic valve interstitial cells through the AKT signaling pathway.
Object: To establish an efficient Bac-to-Bac baculovirus expression system for the expression and purification of the secreted form of varicella-zoster virus (VZV) glycoprotein E (gE) and to evaluate the physical-chemical properties and immunogenicity of gE. Methods: Gibson assembly homologous recombination kit was used for pFastbac-VZV gE recombinant blasmid construction. Expression of the recombinant protein was prepared in baculovirus-infected High-Five TM insect cell after protein expression difference were identified between sequence optimization or non-optimization using sf9 insect cell. ELISA and Western blot were employed for the verification of physicochemical properties of Ni-NTA-exclusion chromatography purified protein gE. Immunogenicity assay of recombinant VZV gE was identified with serum titer determination and immunofluorescence reaction with na?ve VZV. Results: The pFastbac-VZV gE recombinant plasmid was successfully constructed according to PCR and enzyme digestion identification assay. Recombinant bacmids extracted with a BAC/PAC DNA extraction kit were transfected into sf9 insect cell for baculovirus preparation. VZV gE was expressed in baculovirus-infected sf9 cell in a generation-dependent increasing manner according to Western blot assay. Obviously, sequence optimization enhanced gE production, but most of the proteins existed intracellularly. Highly purified gE worked well with VZV monoclone antibody 9C8 by ELISA. High reaction titer of immune serum with gE was identified and immunofluorescence showed interaction between serum and VZV in APER-19 cell. Conclusion: VZV gE recombinant protein highly expressed in Bac-to-Bac baculovirus expression system are generated and laid the foundation for further research and development of VZV subunit vaccine.
In live cells, cyclic adenosine monophosphate (cAMP) was produced from ATP catalyzed by adenylate cyclase and continuous supply of energy and precursor substance was necessary for ATP biosynthesis. The addition of hypoxanthine improved cAMP productivity by 39.1% due to the activated purine salvage pathway when compared with control. However, fermentation ceased at 51h with low cAMP yield and cell concentrations caused by insufficient energy supply. When hypoxanthine and 2g/L-broth (NaPO3)6 were added collaboratively, cAMP concentration (7.24g/L) was improved by 125.5% and 93.5% respectively, compared with those with hypoxanthine and (NaPO3)6 addition individually. The fermentation process with hypoxanthine and (NaPO3)6 coupling addition combined the advantage of salvage pathway and low-polyphosphate which accelerated cAMP biosynthesis and accumulation.
Ammopiptanthus mongolicus is a plant with very strong tolerance to abiotic stresses.To investigate whether AmNAC3,a transcription factor gene from this species,plays roles in tolerating cold and drought, first performed the expression analysis of this gene using semi-quantitative RT-PCR.The results showed that in the seedlings cultivated in normal growth conditions, AmNAC3 had a clear basic expression and the expression level was obviously up-regulated under the drought treatment.In contrast,the expression level of AmNAC3 was weakly up-regulated under the cold stress. Then acquired the 5'-terminal sequence of AmNAC3 by 5'RACE and its full-length cDNA sequence.The whole coding region (846bp) cDNA was cloned by RT-PCR and was inserted into plant expression vector.Transgenic Arabidopsis plants of AmNAC3 was obtained by Agrobacterium mediated genetic transformation.Further analyses showed that the transgenic and wild type plants presented similar drought and cold resistance.However, the water loss rate and stomatal aperture of the detached leaves of transgenic lines were greater than those of wild type.Moreover,the transcript levels of ABI1 and ABI2, two genes related to stomatal closure,were down-regulated in transgenic seedlings.These results indicate that AmNAC3 may primarily function in the response to drought stress and regulation of stomatal closure and leaf water retention but play less role in coping with low temperature stress.
Objective: To construct the vectors of human-mouse chimeric antibody ChAb26 with anti-p185, which can be expression in transgenic mice mammary gland and to express the human-mouse chimeric antibody ChAb26 with anti- p185 by mammary gland bioreactor of transgenic mice. Methods: Firstly, to obtain heavy chain gene H and light chain gene L of human-mouse chimeric ChAb26 by PCR. Secondly, the heavy chain gene H and the light chain gene L of human-mouse chimeric antibody ChAb26 connection to pBC1 after their serial numbers are true by DNA sequencing technology proved. Finally, using DNA sequencing technology for detection the vectors of human-mouse chimeric antibody ChAb26 with anti-p185, pBC1-H and pBC1-L and firstly, the pBC1-H plasmid and pBC1-L plasmid to be linearized by SalI and NotI.After,they become the pBC1-H-linear plasmid and pBC1-L-linear plasmid. Secondly,the pBC1-H-linear plasmid and pBC1-L-linear plasmid to be co-injectioned into the fertilized eggs of FVB mice by microinjection. Thirdly,using mouse tail direct PCR kit for detection the positive clones of the founder mice and their descendants.Furthermore,using RT-PCR and real-time PCR technique for detection the express mRNA of the human-mouse chimeric antibody ChAb26 in transgenic mice mammary gland.The last,using ELISA and Western blot technique for detection the express of mRNA of the human-mouse chimeric antibody ChAb26 in transgenic mice mammary gland. Results: Sequence analysis showed that the serial numbers of pBC1-H plasmid and pBC1-L plasmid are true.Sequence analysis showed that all of 8 founder mice were the positive clones by mouse tail direct PCR identification.Besides,the positive clones rate of their descendants average value is 30%,from F1 to F4 generation.Western blot result showed that a 50kDa heavy chain specific band and a 25kDa light chain specific band were observed in milk of transgenic mice,this meaning milk of transgenic mice containthe human-mouse chimeric antibody ChAb26 with anti- p185. ELISA result indicated that the milk of transgenic mice can combine with anti-human IgG (Fc specific)-HRP antibody produced in goat and anti-human Kappa light chain antibody produced in goat,this meaning milk of transgenic mice containthe human-mouse chimeric antibody ChAb26 with anti- p185.RT-PCR and real-time PCR technique result indicated that the express mRNA of the human-mouse chimeric antibody ChAb26 in transgenic mice mammary gland. Conclusion: The anti-p185 erb B2 human mouse chimeric antibody ChAb26 transgenic animal mammary gland specific expression vectors pBC1-H and pBC1-L were successfully constructed and the anti-p185 erb B2 human mouse chimeric antibody ChAb26 transgenic mouse mammary gland bioreactor model was prepared for future anti-p185 erb B2. The human and mouse chimeric antibody ChAb26 transgenic bovine mammary gland bioreactor laid the theoretical and technical basis.
Site-directed mutagenesis is a common method in molecular biology research. For single-site mutagenesis,the classic method (overlap extension PCR) and the recently developed methods (including rolling circle replication and megaprimer PCR) have both advantages and disadvantages. For multi-site mutagenesis,single-site mutagenesis with multiple rounds PCR is probably still the best strategy although it is tedious and inefficient. In order to improve the efficiency of multi-site mutagenesis,the classic overlap extension PCR can be improved appropriately. First of all,it is necessary to analyze the relative positions between the two mutation sites and develop different mutation primer design strategies accordingly. If two sites are adjacent to each other,or with a distance less than 15bp,one mutational primer pair which contains both mutation sites can be designed and complete the two sites together. If the distance between two sites is between 15-30bp,two sets of mutational primer pair with each set covering each site are necessary,with overlapping regions between these two sets of primer pair to enable second round PCR covering the two mutation sites. If the distance between two mutant sites is further increased,it is implausible to design one mutational primer to cover both mutation sites and use two sets of primer pair with overlapping regions due to the limited length of DNA synthesis in one chemical reaction. Therefore,a new strategy of multi-site mutagenesis by multi-fragment overlap extension PCR was proposed. This multi-fragment strategy divides the gene into three fragments which can be joined together in the second round of PCR in a multi-fragment overlap extension manner with two pairs of mutant primers with overlapping regions. The multi-fragment overlap extension PCR can be adopted when the distance between the two mutation sites is longer than 90bp,considering the limitation of DNA detection by electrophoresis and the efficiency of purification of DNA fragment from agarose gel. Combining the above strategies about mutational primer designing with multi-fragment overlap extension PCR,it is possible to produce multi-site mutations simultaneously in one mutagenesis cycle. Taking the Rb1 gene as an example,its C-terminal fragment contains several potential phosphorylation sites of CDK2/4. A phosphorylation mutant of Rb1 which is designated as PSM6 contains 6 site-directed mutations on phosphorylation sites of C-terminal of Rb1 gene. To achieve PSM6 mutant of RB1 C-terminal fragment,two pairs of mutant primers were designed using the multi-fragment strategy. In the first round PCR,three fragments were amplified using wild type Rb1 gene C-terminal as template,and the PSM6 mutant were amplified using the three fragments overlap extension product from the first round PCR as templates in the second round of PCR. The DNA product,which was supposed to containing 6 phosphorylated site mutants,was digested by restriction endonuclease and then ligated into corresponding plasmid vector by conventional molecular cloning method. According to the DNA sequencing result,it was found that the inserted fragment of recombinant plasmid contains all 6 phosphorylated site mutants as expected. Therefore,a flexible primer design strategy,combined with multi-fragment overlap extension PCR,can be used to produce multi-site gene mutants with one round of mutagenesis,which is a powerful method for multi-site mutagenesis in the research work.
Ketone reductase CgKR2 transforms prochiral carbonyl compounds into high value-added chiral alcohols in one reaction step. Using this enzyme as a biocatalyst could solve the problems of tedious procedures and high costs in the traditional preparation of chiral alcohols, and has a good economic benefit potential. Biocatalysis of OPBE into (R)-HPBE, an important pharmaceutical intermediate of ACEI antihypertensive drugs, is a good application of CgKR2. However, extensive application of CgKR2 was hindered by its complicated and high-cost preparation. Highly efficient secretory expression of CgKR2 was obtained using Brevibacillus choshinensis. With a simple one-step Ni-affinity chromatography, CgKR2 was purified to high purity. The overall yield achieved 7.8mg per liter of fermentation. The purified CgKR2 was characterized in microplate assays, which indicated a specific activity of (78.32±7.62)U/mg, a Km of (0.2±0.02)mmol/L, a Vmax of (117.64±3.6)μmol/(min·mg), and a Kcat of 73s -1. These characterization data were consistent with the data previously reported on CgKR2. Moreover, CgKR2 was obtained ,it can be retained 80% of the activity after 72h of incubation under 30℃, showing a much better stability than the enzyme prepared by previous methods. A simple and highly efficient procedure for CgKR2 expression and purification were developed, which decreased the preparation cost and simplified the process. The new procedure not only would improve the usage of CgKR2 in chiral alcohol preparations, but also sets a good reference for preparation method developments of other biocatalytic enzymes.
To construct ldhL gene deletion strain in Lactobacillus delbrueckii subsp. bulgaricus.Bulgaria lactobacillus CICC21101 was employed as the original strain, amplified the upstream sequence ldhL1 and downstream sequence ldhL2 by PCR, obtained successfully ldhL gene deletion fragment which includes both upstream and downstream sequence, then connected to knockout plasmid pGhost4, electricity to Bulgaria lactobacillus CICC21101, screening at low temperature. The results indicated that the ldhL gene knocked out deficient mutant was obtained.By gene knockout, the yield of D-lactic acid decreased from 30.5g/L to 4.8g/L, the yield of L-lactic acid increased from 25.4g/L to 58.3g/L, and the optical purity increased from 54.56% to 90%. It was a foundation to study the detail functions of ldh in Lactobacillus delbrueckii subsp. bulgaricus,it will lay the foundation of constuction on D-lactic acid engineering bacteria.
Exosomes are naturally occurring biological nanomembranous vesicles (40 to 100 nm) of endocytic origin that are released into the extracellular space from diverse cell types . They have pleiotropic functions such as antigen presentation,intercellular transfer of protein , RNA and lipids. The exosomes secreted by tumor cells play an important role in the physiological and pathological processes which are involved in the occurrence, development and migration of tumors. At present, given the important significance for early diagnosis, for efficacy evaluation and prognosis analysis, the identification for specific markers from tumor exosomes has become the focus of cancer researchers. The recent advances of exosome in basic research as well as its application in cancer diagnosis were reviewed.
KLF8 (Krüppel-like factor 8) is a novel member of the Krüppel-like transcription factor family. KLF8 is widely expressed in different tissues such as skin, adipose, and esophagus, etc. Early studies showed that KLF8 protein was possessed of transcriptional inhibitory activity and played an important role in embryonic development. In recent years, increasing studies indicated that KLF8 was highly expressed ubiquitously in various types of tumor tissues, and acted as a transcriptional activator. KLF8 could promote tumorigenesis process through regulation of cell proliferation, migration, invasion, EMT, and cancer cells stemness. This article will review the research progress of KLF8 in recent years and systematically expound KLF8 expression regulation and its oncogenic mechanism. It may provide a reference for comprehensive understanding and further study of KLF8 in tumorigenesis.
Antimicrobial peptides (AMPs) are produced in various living organisms as first-line host defenses against potential pathogenic microbes in their surroundings. Pioneering studies showed that AMPs can directly interfere with the integrity of the bacterial cell membrane and cell wall though membrane-disruptive mechanism. In addition to interact with membrane, there are increasing evidence to indicate that AMPs have intracellular targets to achieve efficient killing, including nucleic acids binding, inhibiton of protein synthesis and protein-folding, inhibition of cell wall biosynthesis, inhibition of protease and cell division. Due to unique action mechanisms and broad-spectrum of activity, there are continued efforts in exploiting potential applications of AMPs in different area. However, some issues need to be resolved before AMPs be widely used, like AMPs stability, decreased antimicrobial activity and AMPs resistance. In this review, it will be mentioned that current knowledge and recent progress in AMPs action mechanisms, especially non-lytic features, mechanism of AMPs resistance and the potential problems associated with AMPs applications.
In monoclonal antibody drug production process, its glycosylation modification may be affected by a variety of process parameters, therefore come into being heterogeneity easily, and Antibody glycosylation is closely related to antibody half-life, immunogenicity, ADCC, CDC, and so on, so glycosylation is an important quality index for monoclonal antibody drugs, It is necessary to pay attention to and regulate the development of biological drugs, especially in the development of biosimilar. This paper strengthens the understanding of stable and consistent glycosylation by discussing the influence of various important control indicators in the culture process, such as pH, DO, osmolarity and temperature, to guide the efficacy and safety of monoclonal antibody drugs.
The ulvan is consisted of 3-sulfated rhamnose, glucuronic acid, iduronic acid and ran-domly ranged xylose. The polysaccharide and the oligosaccharides degraded from ulvan have been widely used in medical and food industries. In order to promote the utilization of ulvan and ulvan lyase, this review summarized the source, classification, sequence and evolutionary relationship, biochemical characteristics, structure and mechanism of ulvan lyase. Therefore, this review would provide help to the related researchers.
