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Prokaryotic expression method of cotton bollworm midgut serine protease gene

A technology of trypsin gene and intestinal serine, which can be used in genetic engineering, plant genetic improvement, botany equipment and methods, etc., and can solve problems such as unclear mediation of toxin degradation

Inactive Publication Date: 2019-08-02
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, which serine proteases are involved in the activation of the protoxin, which mediate the degradation of the toxin, etc. remain unclear

Method used

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  • Prokaryotic expression method of cotton bollworm midgut serine protease gene
  • Prokaryotic expression method of cotton bollworm midgut serine protease gene
  • Prokaryotic expression method of cotton bollworm midgut serine protease gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 takes the expression of rHaSP3 protein of cotton bollworm as an example

[0046] Step 1: Extract total RNA from the midgut of cotton bollworm larvae, and clone the midgut protease gene fragment of cotton bollworm

[0047] Total RNA was extracted from the midgut of the larvae of the SCD strain of cotton bollworm (the second day after the fifth instar), and the first strand of cDNA was synthesized. The ORF of the hasp3 gene was analyzed. In order to ensure the correct reading mode and the correct termination of the C-terminus, the 5' ends of the upstream and downstream primers were respectively introduced with EcoRI and HindIII restriction sites. Using the midgut cDNA of cotton bollworm as a template, PCR amplification of the hasp3 gene fragment was carried out using 5' end specific primers and 3' end specific primers. The PCR reaction conditions were pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at ...

Embodiment 2

[0068] Embodiment 2 takes the expression of rHaSP6 protein of cotton bollworm as an example

[0069] Step 1: Extract total RNA from the midgut of cotton bollworm larvae, and clone the midgut protease gene fragment of cotton bollworm

[0070] Total RNA was extracted from the midgut of the larvae of the SCD strain of cotton bollworm (the second day after the fifth instar), and the first strand of cDNA was synthesized. The ORF of the hasp6 gene was analyzed. In order to ensure the correct reading mode and the correct termination of the C-terminus, the 5' ends of the upstream and downstream primers were respectively introduced with EcoRI and XhoI restriction sites. Using the midgut cDNA of cotton bollworm as a template, the PCR amplification of the hasp6 gene fragment was carried out using 5' end specific primers and 3' end specific primers. The PCR reaction conditions were pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 64°C for 30 s, extension at...

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Abstract

The invention discloses a prokaryotic expression method of cotton bollworm midgut serine protease gene. The prokaryotic expression method of cotton bollworm midgut serine protease gene comprises the following steps: extracting a total RNA from the midgut of a cotton bollworm larvae, and cloning cotton bollworm midgut protease gene segements; constructing a cotton bollworm midgut protease gene on aPet41 expression vector with His and Gst double tags; expressing, purifying, concentrating, removing the tag of and identifying the recombinant protein. The method directly expresses the mature peptide of cotton bollworm midgut serine protease, so as to avoid the in vitro activation step of zymogen after the expression of the zymogen, and improve the success rate of expressing the bioactive midgut serine protease.

Description

technical field [0001] The invention relates to biotechnology, in particular to a method suitable for prokaryotic expression of midgut serine protease in cotton bollworm and its application. [0002] technical background [0003] Serine proteases (serine proteinases, SPs) are the largest family of hydrolytic enzymes in the biological world, including trypsin, thrombin, chymotrypsin, elastase and so on. They all contain an active center (Ser, His, Asp) composed of three amino acid key residues, and have the same hydrolysis mechanism, but the difference in the residues at the substrate binding pocket determines their respective selectivity to the substrate (Barrett et al. , Handbook of proteolytic enzymes. 2003). Currently, the three-dimensional structures of about 250 serine proteases are known. These proteases are all β proteins, and the core region is mainly composed of two β-fold barrels (ie, the N-terminal domain and the C-terminal domain). Each β-fold barrel is composed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N15/57C12N15/70
CPCC12N9/6408C12N15/70
Inventor 杨亦桦王朦张双双吴益东
Owner NANJING AGRICULTURAL UNIVERSITY
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