In order to prepare the monoclonal antibody of Cancer testis 55 (CT55), the prokaryotic expression plasmid with human CT55 fragment was constructed first, and the plasmid was expressed in the Rosetta competent cell. The target protein was purified and immunized for 6 weeks young female BALB/c mice. According to the preparation method of the conventional monoclonal antibody, the spleen and myeloma cells (sp2/0) of mice was prepared to fuse together. The hybridoma cell lines were obtained by indirect ELISA method and 2 successive subclones, such as 3D8B7B12, 4C8E1C9 and 3D8C10G9. The results of ELISA and Western blot analysis showed that the screened cell lines could produce monoclonal antibodies, and the antibody could specifically recognize the prokaryotic expression and eukaryotic expression of CT55 protein. Specific antibodies can be used in immunofluorescence test, and fluorescence is mainly located at the edge of nucleus. The results showed that monoclonal antibodies against human CT55 protein were successfully prepared. The preparation of CT55 protein monoclonal antibody has laid the material foundation for the rapid etiological diagnosis of cancer of liver cancer, gastric cancer and colon cancer, and the structure and function of CT55 protein.
Objective:To investigate the effect of miR-196a-5p on proliferation and differentiation of mouse adipocyte, and explore its potential molecular mechanisms.Methods:① Utilizing RT-PCR, miR-196a-5p expression levels in adipose tissues from obese or normal mice were measured; ② The miR-196a-5p expression level during preadipocyte differentiation were measured by RT-PCR method, after 3T3-L1 cells were induced to differentiate by cocktail method; ③After miR-196a-5p mimics or inhibitors were transfected into 3T3-L1 cells, CCK8 and EdU detection were performed to evaluate the effect of miR-196a-5p on its proliferation; ④ Measuring the effect of miR-196a-5p on 3T3-L1 cells differentiation by Oil red O staining and triglyceride assay; ⑤ Detecting the effect of miR-196a-5p on the expression levels of 3T3-L1 cells proliferation and differentiation related genes; ⑥ Based on previous reports, using bioinformatics and luciferase reporter assays to identify targets that miR-196a-5p regulates preadipocyte differentiation.Result:①miR-196a-5p not only were highly expressed in adipose tissues of obese mice, but also were expressed dynamically during 3T3-L1 cells differentiation; ②When compared with negative control, mimics transfection inhibited 3T3-L1 cells proliferation, inhibitors transfection promoted its proliferation;③When compared with negative control, mimics or inhibitors transfection increased or decreased lipid accumulation and triglyceride content, respectively; ④When compared with negative control, mimics transfection repressed proliferation related markers (Cyclin D1,Cyclin E,CDK2 and CDK4) and promoted differentiation related markers (PPARγ,C/EBPα,LPL and aP2), however, inhibitors transfection had an opposite effect than that of mimics transfection; ⑤ The miR-196a-5p mimics significantly suppressed a luciferase reporter gene whose expression was regulated by the mouse MAP4K3 and MAPK1 mRNA 3'UTR, whereas mutation of the miR-196a-5p binding site in murine MAP4K3 and MAPK1 3'UTR completely abolished this response.Conclusions:miR-196a-5p might inhibit 3T3-L1 preadipocyte proliferation, and enhance its differentiation. The regulation of preadipocyte differentiation may be mediated by targeting MAP4K3 and MAPK1.
Objective:Based on fluorescence polarization technology, a binding assay targeting the interaction between tomato spotted wilt virus (TSWV) nucleoprotein (NP) and nucleic acid was established and used for drug screening.Methods:The full-length NP DNA amplicon was cloned into the pGEX-6p-1 expression vector, and the construct pGEX-6p-1-NP was transformed into E. coli strain BL(DE3). Recombinant NP protein was purified with a general protocol. A fluorescence polarization assay that is sensitive to inhibitors disrupting TSWV NP/nucleic acid interactions was established. The assay’s DMSO tolerance, incubation time, stability and variability were studied and a pilot drug screening was performed.Results: The recombinant plasmid pGEX-6p-1-NP was successfully constructed and the high quality recombinant NP was purified. A 384-well fluorescence polarization assay targeting TSWV NP and nucleic acid interaction was developed and validated, with a signal-to-noise ratio of 8:1 and a Z factor of 0.82 was obtained, demonstrating the assay is HTS compatible. The assay was used to screen 1 000 compounds in the chemical libraries. After the primary screening, one compound with IC50 of 4.146μmol/L was identified. Conclusion:The fluorescence polarization assay is stable and suitable for the screening inhibitors blocking the interaction between NP and nucleic acids, and the compound provides a reference for the prevention and control of TSWV.
Objective:to establish an efficient method for construction of a phage display library of single chain Fv (scFv) antibody efficiently from mouse immunized with ovalbumin (OVA) and obtain anti-OVA single chain antibody.Methods:Balb/C mice were immunized with OVA. The heavy chain and light chain variable region gene of immunoglobulin were amplified from mRNA of spleen cells of mice with high serum OVA antibody titer by RT-PCR and joined by a DNA linker with seamless cloning. These fragments were inserted into phage expression vector to construct a phage display library. After measurement of library capacity, affinity selection and ELISA analysis were run to find high affinity scFv. Its protein was expressed by Epxi-CHO cells and identified by Western blot analysis.Results:The OVA scFv phage display library was constructed and its library capacity was 1.2 x 10 7 cfu. Eight high affinity scFv were screened from this library. The 2# clone with the highest affinity was cloned into the eukaryotic expression vector and expressed in expi-CHO cells. The Western blot analysis showed that it is a soluble antibody. Conclusion:A highly efficient method for construction of a scFv phage library and generated a high affinity OVA scFv antibody are established. These laid a good foundation for the development of the research of OVA ELISA analysis kit.
The hiTAIL-PCR (high-efficiency thermal asymmetric interlaced PCR) was adopted to study the characteristic of insertion site in genetically modified rice BPL9K-2. As a result, a 450bp fragment of left flanking sequence was discovered. By comparison with rice genome database, the insertion site of exogenous gene located on No. 1037765 of chromosome 10 was found. A 485bp fragment of right flanking sequence was amplified using the primers that were designed according to the sequence of integration site on rice genome and right sequence of exogenous gene. The event-specific PCR detection method was developed based on the left and right flanking sequences, which produced 449bp and 485bp fragment respectively in genetically modified rice BPL9K-2, specifically. The event-specific PCR detection method, with high specificity and sensitivity, could detect the genetically modified ingredients in samples containing 0.1% genomic DNA of BPL9K-2. Based on the flanking sequence, a tri-primer PCR method was developed to identify its genotype of exogenous gene in segregation generation quickly and accurately. The above methods established in this research provide technical supports for the utilization and detection of genetically modified rice BPL9K-2.
A unicellular marine microalgae was isolated at the seaside of Yantai. Both morphological identification and sequence analysis of 18S rDNA showed that this eukaryotic marine microalgae strain is Tetraselmis sp., named 138 algae. A Box-Behnken central composite design was utilized as an optimization of protoplast preparation rate within the experimental range. An equation was obtained to fit the empirical evidence of protoplast formation rate (Y).Y1=88.30-0.19X1+2.44X2+2.28X3-0.95X1X2-3.13X1X3+3.78X2X3-9.85$X_{1}^{2}$-7.95$X_{2}^{2}$-7.22$X_{3}^{2}$. The optimum conditions of protoplasts formation was cellulose: macerozyme at 5:2, concentration of mixed enzymes at 4.5% (2.7% cellulose and 1.8% macerozyme) and buffer pH at 6.4. The theoretical maximum rate is 81.5%. Three parallel verification test proved actual measured rate is 80.5%. The error between model prediction and experimental testing is about 1.24%. Then the transformation efficiency of protoplast was confirmed by the transient expression of GFP gene.
Lipases being important industrial enzymes, which have been widely used in many industrial fields. Compared with free lipases and physical or chemical immobilized lipases, whole-cell lipases have the advantages of simple preparation, no protein extraction and purification, natural immobilization, better stability and resistance, low cost of preparation and equipment, etc. Therefore, the utilization of lipases in the form of whole-cell is known as one of the most promising ways to reduce the cost of biotransformation. The study on whole-cell lipases has always been a hot spot in the lipase field. The research advances in whole-cell lipases, including the wild-type whole-cell lipases and the gene engineered whole-cell lipases, were summarized and reviewed. Furthermore, the future research directions of whole-cell lipases were prospected so as to provide a useful reference for the follow-up studies.
Antibiotics are used to arrest essential bacterial signaling and/or metabolic pathways,causing bacterial cell death.Overuse and misuse of antibiotics have led to dysbacteriosis and drug resistance.The application of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated) systems provides a new method to kill the drug-resistant microbes specifically by designing programmable sequence-specific antimicrobials.Currently,the most efficient CRISPR/Cas systems are type I and type II as a novel antimicrobial tool in selective removal of bacterial pathogens.Bacteriophage has been developed as a delivery vehicle,but the membrane vesicles had more potential for transporting the CRISPR/Cas systems into the targeted resistant pathogen.
Gut microbiota is an important part of human micro-ecology, and also the largest and most complex microbial ecosystem. Gut microbiota were recently proposed to have an important role in the host nutrient absorption, the development of the intestinal immune system, and other important physiological processes; so they are closely related to human health and disease. The exclusion of enteric pathogens by these commensal microbes partially depends upon the production of bioactive compounds such as polyunsaturated fatty acids (PUFAs) et al. At the same time, these fatty acids can be further transformed into polyunsaturated fatty acid derivatives with special structure and function under the action of enteric microorganisms. These key enteric microbial byproducts are critical to maintaining a healthy gut flora. In addition, PUFAs play multiple key roles in host defense and immunity, including anti-inflammation and anti-oxidative activity, as well as the competition of intestinal pathogens. The source of polyunsaturated fatty acids in the intestinal tract and its important physiological functions, and further introduces the transformation and derivation mechanism of intestinal microorganisms to polyunsaturated fatty acids were mainly reviewed. And it pointed out that enteric microorganisms are the production strain seed bank of special polyunsaturated fatty acids and derivatives, so increase the microbial species of functional oils production.
In view of the adverse effects of avian bacterial diseases on the healthy development of the poultry industry, there is an urgent need to develop new sensitive antimicrobial agents that improve bacterial resistance so as to achieve the purpose of treating bacterial diseases. Cyclic peptides are candidates for antibacterials because they have more biological activities and medicinal properties than linear peptides. The discovery of natural antibacterial cyclic peptides, the synthesis of antibacterial cyclic peptides and the application status of antibacterial cyclic peptide drugs were mainly reviewed. It is expected to help readers to further understand the research situation of antibacterial cyclic peptides and provide assistance for the development of new antibacterial cyclic peptide drugs.
Food safety has been the focus of public health throughout the world. Loop-mediated isothermal amplification ( LAMP ) technique is a kind of simple and rapid isothermal nucleic acid amplification technology with high specificity, and outstanding sensitivity. Recently, LAMP has been widely evaluated and applied in the field of nucleic-acid detection. LAMP technology can be applied to the food safety detection field, quickly and accurately monitored in a number of food safety problems harm to human health. Therefore, the advantages of LAMP technology in food safety detection are analyzed and combined with the combination of LAMP technology and new diagnostic technology platform.
As the main component of eucalyptus oil, 1,8-cineole is a monoterpene compound exhibiting a wide range of bioactivities. It has been applied broadly in the fields of pharmaceuticals, food, and cosmetic industries. The commercially available eucalyptus oil is mainly extracted from eucalyptus leaves. The extraction process requires intensive labor and abundant natural resources, and is prone to contaminate the environment. 1,8-cineole will be produced greenly with the rapid development of microbial metabolic engineering and synthetic biology, and with more and more biosynthetic pathways of terpenoids have been deciphered in the future . The biosynthetic pathway of 1,8-cineole, the structures and functions of 1,8-cineole synthases, and the microbial biosynthesis of 1,8-cineole are reviewed. The bottleneck problems encountered in the production of 1,8-cineole with engineered microbial strains are summarized, and the corresponding solving strategies are proposed,that will provide references for the construction of engineered microbial strains with high yield of 1,8-cineole or other monoterpenes.
Objective:To analyze the development status and trend of PD-1/PD-L1 monoclonal antibody in the sense of product manufacturing.Methods:Based on the Cortellis database of Clarivate analytics, the searching results with utilizing quantitative analysis and comparative analysis methods were analyzed.Results:Currently, five PD-1/PD-L1 monoclonal antibodies have been launched into markets, another three PD-1/PD-L1 monoclonal antibodies are at registration phase and four at phase III clinical trial. In addition, business deals related to PD-1/PD-L1 monoclonal antibody are increasing in recent years, including more than 10 deals so far, ranging from drug development, commercial license, and patent assets sales to drug R&D cooperation in early phase. As for China, several PD-1/PD-L1 monoclonal antibodies are at clinical stage, and a bright prospect can be expected for Chinese oligonucleotide therapeutic product markets.Conclusion:Although PD-1/PD-L1 monoclonal antibody market still at its preliminary stage, however, with the continuous development and improvement of future technology, it is believed that more PD-1/PD-L1 monoclonal antibody will be launched in the future, providing a new opportunity for the treatment of cancer and other diseases.
