What secrets do you make in the packaging of viruses?
First, operational safety (1) The operation of lentivirus can be carried out in the biological safety cabinet and the conventional ultra-clean workbench (without opening the exhaust fan); (2) The operator must wear a lab coat, wear a mask, disposable gloves, and guard against venom directly contacting the eyes, ears, or skin wounds; (3) Pay attention to avoid scatter and dripping of venom during operation, and be careful not to scratch the skin with sharp equipment or needles; (4) Inadvertently contact the venom station and the equipment to be recycled, the virus can be inactivated by a solution such as 0.25% SDS, 70% ethanol, 1% sodium hypochlorite; (5) Used work surface, microscope stage, etc., wiped with 70% ethanol; (6) Waste materials such as culture vessels and tips should be immersed in 84 disinfectant (1:9 dilution) or sterilized at 121 °C before disposal. Second, the preservation of lentivirus (1) The lentiviral vector is transported on dry ice and stored at -80 °C for 6 months. When stored for more than 6 months, the working titer needs to be re-explored. (2) The frozen venom is placed on ice or melted at 4 °C before use. It is best to use it on the same day. The unused venom can be temporarily stored at 4 ° C for no more than 2 days. (3) Repeated freezing and thawing will seriously reduce the lentivirus titer, and each freeze-thaw can be reduced by more than 20%. It is recommended to use a small amount of cryopreservation according to the dosage at the initial use. Lentiviral packaging Third, slow virus packaging common problems and prevention 1. Common cell lines that are not suitable for polybrene Polybrene can increase the efficiency of lentiviral infection by neutralizing the charge on the cell surface glycoprotein sialic acid residues. However, it has obvious cytotoxicity to some cells, and if it is used, it will affect the experimental effect. These cells are, for example, HSC-T6 (rat hepatic stellate cells), Raw264.7 (mouse mononuclear macrophage leukemia cells), MCF-7 (human breast cancer cells), H929 (human multiple myeloma cells) ), GBC-SD (human gallbladder cancer cell line), NB4 (human leukemia cell line), kasumi (human leukemia cell line), Jurkat (human leukemia cell line), and the like. It is recommended to consult the corresponding literature reports before starting the experiment, as well as the reference polybrene working concentration. 2 , possible reasons for over-successful expression (1) Plasmid construction has certain problems. (2) Due to the special structure of the sequence, which makes transcription difficult and the mRNA is very unstable, the mRNA level cannot be significantly overexpressed. After excluding the plasmid construction problem, the overexpression of the target gene at the mRNA level was verified by q-PCR, and an easily expressed cell such as 293T was used as a control at the same time. (3) If the mRNA level can be overexpressed, the protein level overexpression may be insignificant due to factors such as post-translational modification, codon composition, translation efficiency, and rapid protein degradation. At the same time, when using WB for protein level analysis, a positive control needs to be set to verify the experimental conditions, especially the antibodies. 3 , how to improve the efficiency of viral infection In addition to improving the MOI, it is possible to try to improve the efficiency of infection from other angles. (1) Appropriately reduce the volume of the incubation medium (the amount of virus added), which can increase the virus density and improve the infection efficiency. The effect on cell status and fluid change time should be considered when reducing the volume of incubation. For example, 96-well plate (50 ul), 24-well plate (250 ul), 6-well plate (1 ml), incubate for 4 h in a small volume, then replenish to normal culture volume, continue to culture for 24 h and then change. (2) Prolong the incubation time appropriately. If you change to 24 h and then change the fluid, pay attention to the need to take into account the state of the cells, mainly for the short incubation time (such as 4-6 h). Because the lentivirus has a half-life of about 8 hours at 37 ° C, the effect of prolonging the incubation time is not obvious. (3) Polybrene can improve the infection efficiency for many cells, but it also has cytotoxicity. It is more toxic to some types of cells (such as neurons and DC cells) and should not be used. When the MOI is lower than 20, there is no need to add Polybrene. The concentration of Polybrene used in specific cells can be reported in the literature and determined by preliminary experiments. Usually in the range of 4-10 ug/ml. Protamine sulfate is less toxic to some cells as a replacement for Polybrene. (4) If there is a flat-angle centrifugal head that can be centrifuged, it can be infected at 2200 rpm for 2 h (37 ° C) while centrifugation, and then continue to incubate in an incubator and culture according to standard conditions. This method is particularly beneficial for infection of suspended cells. (5) If the cell state permits, repeated infections can be performed. After the incubation, the virus-free complete medium was replaced. After 16-24 h of culture, the infection process was repeated 1-2 times to improve the infection efficiency. Fourth, "virus packaging + proteome" exclusive program Option 1: Find downstream action factors based on interest factors After the virus is transfected into the cell, the gene to be studied is overexpressed or knocked down. Through SILAC and iTRAQ technology, we can find the differentially expressed protein in the relevant pathway, and then verify the specific protein of the relevant pathway, and easily find the factor of your research. Key pathway effect factors for related pathways. Option 2: Find specific action factors based on specific conditions Through SILAC and iTRAQ technology, we can screen changes in cell (sample) protein levels under specific conditions (drugs, stimulating factors, etc.) and find relevant proteins for different pathways based on the direction of the research topic. Further, the specific gene is interfered or overexpressed at the cellular level or at the body level, and the cell function or in vivo verification is performed. An organic compound formed by dehydration of amino acids, containing carboxyl and amino groups, and being amphoteric. Also known as "peptide".
Three, is related to the biological body of a variety of cell functions of biological active substances. Hundreds of peptides have been found in living organisms, which are essential participants in the body's complex physiological activities. All cells can synthesize peptides and their functional activities are regulated by peptides. It involves hormones, nerves, cell growth and reproductive fields, and its importance lies in the regulation of various systems, organs and cells in the body. The physiological and pharmacological effects of enzymatic polypeptides are mainly to activate related enzymes in vivo, promote the permeability of intermediate metabolic membrane, or affect specific protein synthesis by controlling DNA transcription or translation, and eventually produce specific physiological effects or play their pharmacological effects. Peptide is better than amino acid, one is faster than amino acid absorption; Second, it is absorbed by the body in a complete form; Third, active absorption (amino acids are passive absorption); Fourth, low consumption, compared with amino acids, peptide absorption has the characteristics of low consumption or no energy consumption, peptide absorption through the duodenum, directly into the blood circulation, their own energy nutrition transported to various parts of the human body; Fifth, peptide absorption is less than amino acid, with the characteristics of unsaturated; Sixth, there are only 20 kinds of amino acids, with countable functions, while peptides take amino acids as substrates, which can be synthesized into hundreds of thousands of kinds.
It is mainly divided into medical polypeptide drugs, peptide antibiotics, vaccines, antimicrobial peptides for agricultural use, small peptides for feed, cosmetics for daily use, soybean polypeptides for food, corn polypeptides, yeast polypeptides and sea cucumber peptides. buy peptides,peptides benefits,peptides bpc 157,peptides buy,peptides cjc 1295 Shaanxi YXchuang Biotechnology Co., Ltd , https://www.peptide-nootropic.com
Two, one of the amide. It consists of two or more amino acids joined by the amino group of one amino acid to the carboxyl group of the other. An amino acid cannot be called a peptide, nor can a peptide be synthesized, but must be a compound of two or more amino acids linked by a peptide bond. Compounds where two amino acids are linked by a peptide bond are called dipeptides; A compound with three amino acids linked by peptide bonds is called a tripeptide, and so on, a compound with thirty-four amino acids linked by peptide bonds is called a thirty-four peptide.
A peptide is a chain of polymers of amino acids
A peptide is a chain of polymers of amino acids
From the functional point of view, can be divided into antihypertensive peptide, antioxidant peptide, cholesterol lowering peptide, opioid active peptide, high F value oligopeptide, food strong taste peptide and so on.
Active peptide, and nutrition, hormone, enzyme inhibition, regulation of immunity, antibacterial, antiviral, antioxidant has a very close relationship. Polypeptides are generally divided into: polypeptide drugs and polypeptide health products. Traditional peptide drugs are mainly peptide hormones. The development of peptide drugs has been developed into various fields of disease prevention and treatment, especially in the following fields. [2]
Antitumor polypeptide
Tumorigenesis is the result of many factors, but ultimately all involve the regulation of oncogene expression. In 2013, many tumor-related genes and regulatory factors have been discovered. Screening peptides specifically bound to these genes and regulatory factors has become a new hot spot in the search for anticancer drugs. For example, somatostatin has been used to treat endocrine tumors of the digestive system; American scholars found a hexapeptide can significantly inhibit adenocarcinoma in vivo; Swiss scientists have discovered an octapeptide that induces apoptosis in tumor cells. [2]
Antiviral polypeptide
By binding to specific receptors on host cells, viruses adsorb cells and rely on their own specific proteases for protein processing and nucleic acid replication. Therefore, peptides binding to host cell receptors or active sites such as viral proteases can be selected from the peptide library for antiviral therapy. In 2013, many small peptides with antiviral properties have been screened from peptide banks in Canada, Italy and other countries, and some small peptides have entered the stage of clinical trials. In June 2004, the Chinese Academy of Sciences institute of Microbiology came out of the news, which was undertaken by the Chinese Academy of Sciences known