A kind of beet armyworm vitellogenin receptor, its specific peptide chain, carrier, bacterial strain and application
A technology of vitellogenin, beet armyworm, applied in the field of agricultural science
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0036] Embodiment 1: Obtaining of SEVgR full-length gene
[0037] 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, 5μl MMlv Buffer, 5μl 10mM dNTP, 40U RNasin, 1μl MMlv reverse transcriptase (reverse transcriptase), add ddH 2 O to 25 μl; PCR reaction parameters: 42°C for 1h, 95°C for 10min. According to the conserved sequence of the known insect VgR gene, primers SEVgR-3396F (5'-CGAAGAGGACTGTGATGAACCT-3') and SEVgR-4845R (5'-GACAAATGTGTTGGCGTCAA-3') suitable for PCR amplification were designed to obtain the conserved sequence of the SEVgR 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...
Embodiment 2
[0039] Example 2: SEVgR-specific peptide chain cDNA cloning
[0040] According to the measured full-length sequence of SEVgR gene, the cDNA sequence of SEVgR-specific peptide chain was predicted by DNAMAN software, and PCR amplification primers SEVgR-3F (5′-TGTCATGAAACTGAGTTCATGTG-3′) and SEVgR-3R (5′-GCATGAGAACTGATCCTTGGACG- 3′) to obtain the SEVgR 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 Takara LA DNA polymerase, 1 μl 10 μM upstream primer SEVgR-3F, 1 μl 10 μM downstream primer SEVgR-3R, 1 μl cDNA template prepared in Example 1, add ddH 2 O to 50 μl; PCR reaction parameters are: 94°C for 5 min; 94°C for 30s, 55°C for 30s, 72°C for 1 min, 35 cycles; 72°C for 10min extension. The electrophoresis results showed that after PCR amplification and splicing, a band consistent with the expected size of 786 bp was obtained ( image 3 ). The PCR product was separated by 1.2% agarose gel el...
Embodiment 3
[0042] Example 3: Construction of SEVgR-specific peptide chain DNA prokaryotic expression vector
[0043] The construction method of the prokaryotic expression vector containing the SEVgR-specific peptide chain gene T vector is as follows: the PCR product sequenced correctly in Example 2 is connected to the expression vector pET15B through the cloning sites Nde I and Xho I, and it is verified by enzyme digestion whether a recombinant plasmid is formed. The digestion system was as follows: 0.25 μl of each of the two restriction enzymes (NdeI and XbaI), 1.0 μl of 10× Buffer, 3 μl of plasmid, and ddH 2 O supplemented to 10 μl, water bath at 37°C for 3h. See the enzyme cleavage map after successful ligation Figure 4 . The correctly identified recombinant plasmid named pET15B-SEVgR was transformed into 100 μl E. coli Arctic Express (Zhongding Biotechnology) competent cells, placed on ice for 20 min, heat-shocked at 42°C for 90 s, quickly placed in ice for 5 min, and added 600 μl...
PUM
| Property | Measurement | Unit |
|---|---|---|
| molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap