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Preparation method and application of recombinant Vip Aa protein

A protein and recombinant strain technology, which is applied in the field of preparation of recombinant Vip3Aa protein, can solve the problems that have not yet been reported on the application of Vip3Aa protein in cockroaches, and achieve good agricultural application prospects, high purity, and improved production efficiency.

Inactive Publication Date: 2018-11-16
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there is no report on the application of Vip3 Aa protein in cockroach control

Method used

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  • Preparation method and application of recombinant Vip Aa protein
  • Preparation method and application of recombinant Vip Aa protein
  • Preparation method and application of recombinant Vip Aa protein

Examples

Experimental program
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experiment example 1

[0050] Experimental Example 1 The insecticidal activity of Vip3 Aa protein of the present invention to Periplaneta americana and Blattella germanica

[0051] Take 2mg / ml, 0.5mg / ml, 0.125mg / ml and 0.03125mg / ml Vip3 Aa protein to make 1ml solution, divide into 5 groups, each group has 15 German cockroaches, put German cockroaches into the bacterial culture dish , add 1ml of drug solution, drink water freely, respectively in 12h, 24h and 36h and 48h, observe the mortality rate of German cockroach.

[0052] Take 2mg / ml, 0.5mg / ml, 0.125mg / ml and 0.03125mg / m concentration of Vip3 Aa protein to make 1ml solution, divide into 5 groups, 10 American cockroaches in each group, put the American cockroach into the Erlenmeyer flask , add 1ml of drug solution, drink water freely, observe the mortality rate of Periplaneta americana at 12h, 24h and 36h respectively.

[0053] 3.3.1 Analysis of the activity of Vip3 Aa protein against cockroaches

[0054] The 12h, 24h and 36h LD50 of Vip3 Aa to...

experiment example 2V

[0059] Experimental example 2 Effect of Vip3 Aa protein on the digestive system of Periplaneta americana

[0060] (A) HE staining

[0061] (1) Anesthesia and dissection: place the cockroach on ice for anesthesia, insert an insect needle into the neck of the cockroach and fix it, remove the wings and feet of the cockroach, cut off the body wall from the edge of the abdomen with ophthalmic scissors, and remove excess abdominal cavity with small forceps Straighten the digestive tract, cut along the top of the pharynx and the end of the rectum to obtain the complete digestive system of the cockroach.

[0062] (2) Material collection: the crop, midgut, and columnar colon were cut with a surgical blade, and the sample tissue was smaller than 0.5cm×0.5cm×0.1cm.

[0063] (3) Fixation and embedding: fix with 4% PFA (0.01MPBS, pH7.4), pass through 70% ethanol for 60 minutes, 85% ethanol for 60 minutes, 95% ethanol for 60 minutes, 100% ethanol for 50 minutes twice, Twice in xylene for ...

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Abstract

The invention relates to a preparation method and an application of a recombinant Vip3 Aa protein, and belongs to the field of biological control. The preparation method of the recombinant Vip3 Aa protein comprises the following steps: carrying out a PCR reaction on Vip3 Aa DNA, carrying out double enzyme digestion by using restriction endonucleases BamH I and Xho I to obtain a target gene, linking the target gene with pET21b to obtain a Vip3 Aa / pET21b expression vector, transferring the vector into an E. coil BL21 competent cell to obtain a recombinant strain, and adding IPTG after the recombinant strain grows to a logarithmic growth phase in order to induce expression in order to prepare the Vip3 Aa protein. The protein has a good biological control effect on German cockroaches and American cockroaches, and is suitable for being agriculturally promoted and applied.

Description

technical field [0001] The present invention relates to a method for preparing recombinant Vip3 Aa protein and its application, in particular to a Vip3 Aa protein prepared by induced expression of Vip3 Aa / pET21b / BL21 recombinant strain and its application in the control of German cockroach and American cockroach, belonging to biological field of prevention and control. Background technique [0002] Bt strains can synthesize an insecticidal protein during the vegetative growth stage, and the insecticidal protein is called vegetative insecticidal proteins (Vip). Vips are mainly divided into vip1, vip2 and vip3, and different vips proteins have different insecticidal activities. Vip3 protein is currently the most studied Vip protein. The Vip3 Aa protein has high insecticidal activity against crop pests such as cotton bollworm, beet armyworm, fall armyworm, tobacco armyworm, diamondback moth, Spodoptera litura and cutworm. The action process is as follows: Vip3 protein is hyd...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12P21/02C07K14/325C07K1/14A01N47/44A01P7/04
CPCA01N47/44C07K14/325C12N15/66C12N15/70
Inventor 朱家勇刘文彬金小宝卢雪梅
Owner GUANGDONG PHARMA UNIV
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