Anti-mullerian hormone AMH gene for epinephelus lanceolatus, encoding protein and application of anti-mullerian hormone AMH gene
A technique of Mullerian hormone and saddle grouper, applied in the field of genetic engineering, can solve problems such as the slim chance of catching wild male broodstock
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Embodiment 1
[0056] Example 1. Cloning of saddle grouper anti-Müllerian hormone AMH
[0057] 1. Extraction of total RNA from gonads of saddle grouper
[0058] Take healthy whole grouper fish, anesthetize it with ice bath for about 2 minutes, kill the fish and take samples, separate the gonad tissue, use Trizol reagent to extract the total RNA of gonad of grouper saddle, and its OD 260 / 280 = 1.90.
[0059] 2. Synthesis of the first strand of cDNA
[0060] Get 1 μ g of saddle grouper gonad total RNA sample and carry out DNase treatment to remove the contamination of genomic DNA, mix with RNAOligo dT (sequence shown in SEQ ID NO:3, specifically ttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt) is mixed with RNAOligo dT, and the resulting product is placed in Store at -20°C for later use.
[0061] 3. Cloning of complete cDNA sequence of saddle grouper AMH gene
[0062] According to the information in the saddle grouper tran...
Embodiment 2
[0068] Example 2 Expression and plasmid coating of saddle grouper anti-Müllerian hormone AMH recombinant protein
[0069] 1. Construction of recombinant expression plasmids
[0070]Using the pGEM-T plasmid containing the AMH coding gene as a template, a pair of upstream primers SEQ ID NO: 6 (ccggaattcctgcaggtctcacatggaaagc) containing the EcoRI cleavage site and a downstream primer SEQ ID NO: 7 (ccgctcgagtaacggcatccaacactccttcgcc) containing the XhoI cleavage site were designed. PCR amplification obtained a single band with a product size of about 1497bp, and the electrophoresis results were as follows: figure 1 .
[0071] The mature peptide coding sequence of saddle grouper anti-Müllerian hormone AMH was cloned into the prokaryotic expression vector pET32a to obtain the recombinant expression vector pET32a-AMH (the construction process was as follows figure 2 shown). The exogenous gene sequence in the expression vector was confirmed to be correct by sequencing.
[0072] ...
Embodiment 3
[0082] Example 3 Preparation of saddle grouper anti-Müllerian hormone AMH antibody
[0083] The recombinant protein AMH obtained in Example 2 was purified, and the recombinant protein rabbit polyclonal antibody was prepared, and the immunization steps were as follows:
[0084] 1) Dissolve 1 mg of the recovered recombinant protein in PBS buffer, mix and emulsify thoroughly with an equal volume of complete Freund's adjuvant.
[0085] 2) Four male New Zealand white rabbits aged 1-3 weeks were selected, and 0.5 mL of blood was collected before immunization as a sample control. Complete Freund's adjuvant (1:1) was mixed for the first immunization, 0.5mL antigen solution was added to 0.5mL Freund's complete adjuvant, and the emulsion was sucked with a 2mL syringe to eliminate air bubbles in the syringe.
[0086] 3) The immunization dose of each rabbit is 1.5 mL subcutaneous injection. After the first immunization, the rats were boosted with incomplete Freund's adjuvant (IFA) at th...
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