Vitellogenin, its specific peptide chain, carrier, bacterial strain and application
A vitellogenin and green Lygus technology, applied in the field of agricultural science, can solve problems such as lack of protein expression detection methods
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Embodiment 1
[0043] Embodiment 1: the acquisition of AlVg full-length gene
[0044]The TRIzol method was used to extract the total RNA of Lygus japonica, and the total RNA samples with good electrophoretic patterns and OD260 / OD280 values between 1.8 and 2.0 were selected and combined for mRNA purification, and the first-strand cDNA was synthesized by reverse transcription. The PCR reaction system was: 0.5 μg total RNA, 0.5μl Oligo-dT18, 2μl 5× reaction buffer, 2μl 10mM dNTP, 40U RNasin, 200USuperscriptⅡ, add ddH 2 0 to 10 μl; PCR reaction parameters: 42°C for 30 min, 75°C for 30 min, 4°C for 5 min. According to the known conserved sequence of insect Vg gene, primers AlVg-1F (5'-CGTTGGTAGCCGCATGAAC-3') and AlVg-1R (5'-GAACTGCTGCTGGAACTGCTG-3') suitable for PCR amplification were designed to obtain the conserved sequence of AlVg gene. For the sequence, refer to SEQ ID NO: 19 in the sequence listing. The PCR reaction system is: 2.5 μl 10× reaction buffer, 2 μl 25 mM Mg 2+ , 2 μl 10 mM dNT...
Embodiment 2
[0046] Embodiment 2: AlVg protein-specific peptide chain cDNA clone
[0047] According to the full-length sequence of the AlVg gene in the National Center for Biotechnology Information (NCBI), an authoritative international gene database, the cDNA sequence of the specific peptide chain of the AlVg protein is predicted, the base is shown in SEQ ID NO: 3, the amino acid sequence is shown in SEQ ID NO: 4, and PCR is designed Amplify primers AlVg-3F (5′-CATATGCAAATCCAAATCTCAACGT-3′) and AlVg-3R (5′-TCTAGATCAAGCTTTGCAGCGAG-3′) to obtain AlVg protein specific peptide chain gene sequence, PCR reaction system: 2.5 μl 10× reaction buffer Solution, 2μl 25mM Mg 2+ , 2 μl 10 mM dNTP, 0.5 μl Taq plus DNA polymerase, 1 μl 10 μM upstream primer AlVg-1-F, 1 μl 10 μM downstream primer AlVg-1-R, 1 μl cDNA template, add ddH 2 O to 25 μl; PCR reaction parameters: 94°C for 5 minutes; 94°C for 30s, 60.5°C for 30s, 72°C for 60s, 40 cycles; 72°C for 10 minutes. The results of electrophoresis showed...
Embodiment 3
[0049] Embodiment 3: Construction of AlVg protein-specific peptide chain DNA prokaryotic expression vector
[0050] The method for constructing the prokaryotic expression vector containing the AlVg protein-specific peptide chain gene T vector is as follows: the above-mentioned sequencing is correctly connected into the pGEM-TEasy vector and pCzn1 are both digested with restriction endonucleases NdeI and XbaI, and connected. The enzyme digestion system is: 1.0 μl of each of the two restriction enzymes, 2.0 μl of 10×Buffer buffer, 10.0 μl of gene fragments with appropriate restriction sites, and ddH 2 Make up O to 20 μl, and bathe in water at 37°C for 3h. The ligation system is: 5.0 μl of the recovered vector after enzyme digestion, 10.0 μl of the gene fragment, 1.0 μl of T4 DNA ligase, 2.0 μl of 10×Buffer, and ddH 2 Make up O to 20 μl, react at 16°C for 12-16 hours, and see the enzyme digestion map after successful ligation Figure 4 . After the ligation product was transfor...
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