Product Introduction IPTG is a highly potent inducer that is widely used in laboratories due to its stability during the induction process, as it is not metabolized by bacteria. IPTG is commonly used as an inducer in biopharmaceuticals and enzyme catalysis technology. Application of IPTG Bees play an important role in maintaining natural ecological balance and ecological agriculture, as well as providing high nutrition and health function bee products to humans. However, in the past decade, due to various reasons, a large number of bees have been lost around the world, seriously threatening the beekeeping industry, ecological balance, and rural agricultural production in various countries. The virus that bee larvae are infected with is mainly Saccharid Larvae Virus (SBV). When SBV infects Chinese honeybees, it is called the Chinese Bee Saccharid Larvae Virus (CSBV), which causes incomplete pupation of the Chinese Bee larvae and fills them with viral particles. When the damage is severe, a large number of larvae will die due to infection. At the same time, adults who clean up dead larvae to maintain the living environment of the bee colony will also be infected with CSBV, ultimately leading to the extinction of the population and irreversible losses to the beekeeping industry. In order to detect the changes in ceramide content in the body of Chinese bee larvae before and after infection with CSBV, the ceramide content in the body of Chinese bee larvae infected with CSBV virus was detected. At present, there are mainly test kits for detecting insect ceramides on the market, but there are no test kits for detecting ceramide content in Chinese bees infected with CSBV. Therefore, CN117924437A patent has developed a detection kit and detection method for the virus of Chinese wasp cystic larvae. The specific preparation method for the detection test antibody is as follows: (1) Using neutral sphingophospholipase (AcnSase) from Chinese honeybees as the key enzyme; (2) Using the cDNA of Chinese honeybee larvae as a template, the AcnSMase gene was amplified to obtain a nucleic acid fragment of about 840bp. The target fragment was cut and recovered, connected to the PMD19-T cloning vector and transformed. Single colony was selected for bacterial liquid PCR, plasmid PCR, and double enzyme digestion triple validation. PMD19-T-AcnSMase was successfully constructed, and the next step of expression vector construction was carried out. (3) Recombinant plasmid and empty GST vector plasmid were double digested and the target fragment was recovered. After connecting and transforming, a single colony was selected for bacterial liquid PCR. Plasmid PCR and double digestion verification were successful, indicating the successful construction of the recombinant expression vector. Subsequently, the recombinant plasmid was transferred into Rosetta competent cells, and a single colony was selected for bacterial liquid PCR screening to identify positive strains for subsequent induction of recombinant protein expression. (4) Cultivate the expression strain at 37℃ for 7 hours, and induce it with IPTG with a final concentration of 0.1mM at an OD value of 0.6-0.8 for 8 hours. Perform SDS-PAGE electrophoresis analysis, and there is a significant difference in bands around 58KD, indicating successful induction of the recombinant protein. Detection of the expression form of the recombinant protein revealed that the recombinant protein mainly exists in the form of inclusion bodies in the precipitate; Subsequently, a large number of recombinant proteins were induced and purified using nickel column affinity chromatography for subsequent immunization of mice to obtain polyclonal antibodies. The detection kit in this patent is used to detect the infection of bee pathogenic microorganisms, assist in bee production and related experimental research. Compared with existing insect ceramide detection kits, the cost of this kit is lower.
References CN117929706A A detection reagent kit and detection method for Chinese bee cystic larval virus.
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