Spodoptera exigua vitellogenin (SEVg) as well as specific peptide chain, carrier, strain and application thereof
A technology for vitellogenin and Spodoptera exigua, which is applied in the field of agricultural science, can solve the problems of difficulty in synthesizing specific antibodies, lack of detection methods for protein expression, and difficulty in full-length expression.
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Embodiment 1
[0041] Embodiment 1: Obtaining of SEVg full-length gene
[0042] The total RNA of beet armyworm was extracted by TRIzol method, and the total RNA samples with good electrophoretic pattern and OD260 / OD280 value 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, 2.5μl Oligo-dT18, 5ul MMlv Buffer, 5μl 10mM dNTP, 40U RNasin, 0.5ul MMlv reverse transcriptase (reverse transcriptase), add ddH 2 O to 20 μl; PCR reaction parameters: 42°C for 1h, 95°C for 10min. According to the conserved sequence of the known insect Vg gene, primers SEVg-1F (5'-GCTGAATGGCGACGAGAGCT-3') and SEVg-1R (5'-CTCATGGAAGCGATAATGCGGAC-3') suitable for PCR amplification were designed to obtain the conserved sequence of the SEVg gene. For the sequence, refer to SEQ ID NO: 11 in the sequence listing, the PCR reaction system is: 5 μl 10× reaction buffer, 5 μl 25mM Mg 2+, 8μl 10mM dN...
Embodiment 2
[0044] Embodiment 2: SEVg protein-specific peptide chain cDNA clone
[0045] According to the measured full-length sequence of SEVg gene, use DNAMAN software to predict the cDNA sequence of SEVg protein-specific peptide chain, and design PCR amplification primers SEVg-3F (5'-CATGGCAAACTGGCAAACTTTA-3') and SEVg-3R (5'-TAATTCCACTCTTGATAGCAGAAC -3'), to obtain the SEVg protein-specific peptide chain gene sequence, the PCR reaction system is: 5 μl 10× reaction buffer, 5 μl 25mM Mg 2+ , 8 μl 10 mM dNTP, 0.5 μl Takala LA DNA polymerase, 1 μl 10 μM upstream primer SEVg-3F, 1 μl 10 μM downstream primer SEVg-3R, 1 μl cDNA template, add ddH 2 0 to 0 μl; PCR reaction parameters: 94°C for 5min; 35 cycles of 94°C for 30s, 55°C for 30s, 72°C for 2min; 72°C for 10min. Electrophoresis results showed that after PCR amplification and splicing, a band consistent with the expected size of 2091bp was obtained ( figure 2 ). The PCR product was separated by 1.2% agarose gel electrophoresis and g...
Embodiment 3
[0047] Example 3: Construction of SEVg protein-specific peptide chain DNA prokaryotic expression vector
[0048] The method of constructing the prokaryotic expression vector containing the SEVg protein-specific peptide chain gene T vector is as follows: connect the PCR product with the correct sequencing above into the pGEM-TEasy vector and pCzn1 with restriction endonucleases NdeI and XbaI for double digestion and connection . 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 image 3 . After the ligation product was tran...
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