The Escherichia coli (E.coli) expression system was adopted earlier and is also currently familar with protein expression system. As a host bacterium for recombinant protein production, escherichia coli not only has the advantages of clear genetic background, simple culture operation, high conversion and transduction efficiency, fast growth and reproduction, low cost, but also can rapidly produce a target protein in a large specification, and the level of expressing a heterologous protein product is much higher than that of other protein expression systems. Therefore, Escherichia coli is currently one of the most widely used protein expression systems.
1. We can provide a complete set of solutions for that soluble expression of recombinant protein in E.coli. When the recombinant protein is expressed in large quantities in E.coli, insoluble inclusion bodies are easily formed in cell, which brings troubles for later purification and renaturation.The protein obtained by renaturation has low activity or even no activity. We can improve the expression yield and solubility of recombinant proteins in cytoplasm by selecting appropriate host cells, molecular chaperone co-expression or fusion tag expression, or prepare high-activity recombinant proteins by selecting secretory expression, with a complete set of solutions.
2. E. coli expression system has been widely used, has successfully expressed molecular enzymes, biochemical enzymes, cytokines, antigens, antibodies, toxic proteins or small molecule polypeptides, etc., and has accumulated rich experience in prokaryotic expression system.
Mammalian cell expression system can provide complete post-translational modifications (such as glycosylation and phosphorylation) closer to the natural state for recombinant human proteins, and the protein folding and polymerization close to the natural protein will be formed during protein expression, possessing the necessary spatial structure and modifications for the active protein. The exogenous protein produced by translational reprocessing modification in mammalian cells was more active than other expression systems, and closer to the natural protein. Wuhan Huamei Biotech Co.,LTD.◎Industrial Raw Materials Division has established a mature mammalian protein expression system and purification service platform, providing the expression and purification services of the expression system.
1. The expression vector can be expressed in mammalian cells with high yield and high activity.
2. A variety of expression systems are available: transient expression, stable cell line expression, serum-free suspension expression, large-scale suspended cell expression, etc.
3. The transient expression system uses HEK293 cells and CHO cells as expression hosts, which is easy to transfect with high transfection efficiency, and can provide flexible services to meet the needs of different customers.
4. The steady-state transformation expression system mainly uses CHO cells as the expression host, and is able to have relatively fast growth rate and high protein expression yield in suspension culture. It is the main production cell line.
5. According to the different needs of customers, the use of high standard bioreactor can provide 10L/20L/50L，etc.
6. We have advanced AKTA purifier system, ion column and affinity chromatography column, etc.
Hybridoma antibody technology of mouse is to fuse mouse myeloma cells with immunized mouse B lymphocytes through physical, chemical or biological methods,which forms hybridoma cells that have the characteristics of both parents and secreting homogeneous high specific antibodies against a single epitope of immunogen. The fused cells have the characteristic of infinite reproduction of tumor cells as well as the ability of lymphocytes to secrete specific antibodies or factors. Wuhan Huamei Biotech Co.,LTD.◎Industrial Raw Materials Division has established a mature technology platform with hybridoma fusion screening, evaluation, production,and purification, that provides high-quality diagnostic antibody raw materials to customers.
1. After years of development, the process of Technical operation is fully transparency and well recognized.
2. Supernatant or antibody of hybridoma cell can be screened directly against specific epitopes by indirect competition method, and the selected monoclonal antibodies are highly specific.
3. The all selected hybridoma cells can adapt to the serum-free medium environment, which is easy to scale up and control between batches.
4. According to the different needs of customers, the use of WAVE bioreactor can easily meet the supply demand of customers above 10g level.
5. We possess the cutting edge of AKTA Pure150M chromatography system, and different sizes chromatography columns of ion, affinity, molecular sieve.
The single B antibody platform is an emerging antibody technology which has a short preparation cycle and can obtain the monoclonal antibody with the heavy and light chains naturally paired without cell fusion or gene synthesis. Briefly, a single B antibody platform comprises the following steps:
1. Preliminary extraction of immune cells and isolation of peripheral blood mononuclear cells (PBMC)；) from peripheral blood or immune organs;
2. Specific B cells are identified and separated using specific cell separation technology, magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS) technology;
3. Performing single cell culture on the separated b cells, detecting the antibody content in the culture medium supernatant, and labeling positive cells capable of secreting specific antibodies;
4. Extracting and amplifying antibody genes of positive cells;
5. Clone that antibody gene into an expression vector and expressing in a eukaryotic system;
6. Purification the antibodise has been produced, and evaluating the antibody with the luminescent platform.
1. Rabbit monoclonal antibodies can be obtained by using the single B cell technology platform. Compared with mouse monoclonal antibodies, rabbit monoclonal antibodies have higher affinity and more abundant epitopes.
2. The preparation period is short. The single B cell is only need to be screened and culture in 15 days, and that cell culture time is greatly shortened.
3. The heavy and light chains are naturally paired, and high-affinity antibodies are more easily obtained without pairing and elutriation in vitro.
4. Recombinant expression of antibody genes can preserve the antibodies information more stably and avoid the difference between lots.
5. The invention patent has been applied for the specific cell separation technology, which can improve the single B cell screening positive rate by more than one time.
The Platform of Application Detection
Lipid Emulsion Turbidimetric Immunoassay Analysis (LETIA)
Principle: The specific binding reaction will occur when the antigen to be tested in the sample meets the antibody in the reagent.sequentially, there forms a network of antigen-antibody molecular complex to cause the change of turbidity in the solution. The concentration of the antigen to be tested in the sample can be determined through testing the change of absorbance in the solution.
Advantages: Lipid Emulsion Turbidimetric Immunoassay Analysis has been accumulated for more than 10 years since 2009, that is currently one of the central verification technologies of Wuhan Huamei Biotech Co.,LTD.◎Industrial Raw Materials Division. Based on the specific reaction of antigen and antibody and the principle of latex enhancement and amplification, it has advantages of high sensitivity, good specificity , strong anti-interference ability and so on.
Enzyme-Linked Immunosorbent Assay (ELISA)
Principle: The ELISA technique uses a microplate as the solid phase carrier, and the antigen or antibody labeled with HRP reacts specifically with the antigen or antibody on the solid phase carrier to form a complex, which is quantitatively or qualitatively analyzed by TMB colorimetry.
Advantages: ELISA is based on the antigen-antibody reaction and the efficient catalytic effect of enzymes, which has positives for high sensitivity and specificity. This technology is the most advantageous technical platform of CUSABIO, which has been continuously accumulated since 2007 and plays a significant role. Its application ranges from detection products of scientific research to the screening and identification of diagnostic reagents raw materials, that is currently the foundational platform of detection technology for Industrial Raw Materials Division Diagnostic Reagents Immunoanalysis.
Chemiluminescent Immunoassay Analysis (CLIA)
Principle: The tracer (enzyme, luminescent group) labels antibody , chemical reaction occurs and photon is emitted under the action of a specific substrate.The change of the photon count is corresponding to the substance to be measured in the sample, thereby,the concentration of the substance to be measured can determined in the sample.
Advantages: Tubular particle Chemiluminescent Analysis uses suspended magnetic particles as the carrier, which has higher specific surface area, superior sensitivity, wider linear range, faster detection speed and better repeatability, and is easy to automatical noperate. In Industrial Raw Materials Division, the platform has been applied in the development and application evaluation of diagnostic reagent raw materials since 2014. It has evolved from enzymatic luminescence of template to micro-particle enzymatic luminescence and direct luminescence, that is considered as one of the main platforms for diagnostic applications.
Time-resolved fluorescence lateral flow immunochromatographic assay (TRF-LFIA)
Principle: The antigen to be tested in the sample combines with colloidal gold or fluorescent microspheres with specific antibodies, and then forms a double antibody sandwich complex with specific antibodies fixed on nitrocellulose membrane.The specific immunoassay analysis will be realized by detecting the signal of colloidal gold or fluorescent microspheres.
Advantages: The colloidal gold immunochromatographic technology contains the virtues of simple operation, fast detection and strong portability, which plays an important role in the field of real-time and on-site detection .Beyond that, fluorescent quantitative immunochromatographic technology also realizes quantitative,rapid,ultra-sensitive and accurate trace detection.Industrial Raw Materials Division has accumulated platform technologies based on early POCT diagnosis application and micromolecule rapid detection since 2015, and has improved and perfected the platforms of colloidal gold immunochromatographic technology as well as time-resolved fluorescent immunochromatographic technology, which will focus on the development of analytical technologies for rapid diagnosis of raw material evaluation in Industrial Raw Materials Division.