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Method for producing cobra CT and PLA2 in baculovirus-insect expression system

A technology of insect expression and baculovirus, applied in the field of genetic engineering, can solve problems affecting the function of CT and PLA2 proteins

Inactive Publication Date: 2014-05-21
南宁培元基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Cobra ( Naja ) species all contain CT and PLA2 proteins, but CT and PLA in different species or subspecies 2 There are nucleotide or amino acid differences that may affect the function of CT and PLA2 proteins

Method used

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  • Method for producing cobra CT and PLA2 in baculovirus-insect expression system
  • Method for producing cobra CT and PLA2 in baculovirus-insect expression system
  • Method for producing cobra CT and PLA2 in baculovirus-insect expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0204] Embodiment 1: Guangxi Cobra CT and PLA 2 Obtaining the mature peptide sequence

[0205] 1. Extraction of total RNAR from snake venom glands

[0206] The specific steps of Guangxi cobra venom total RNA extraction are as follows:

[0207] (1) Homogenization treatment Put 50-100 mg of Guangxi cobra venom glands into a sterilized and pre-cooled mortar with liquid nitrogen, add an appropriate amount of liquid nitrogen, grind the tissue fully with a grinding pestle, add the lysate RZ after grinding, The sample volume should not exceed one-tenth of the RZ volume of the lysate.

[0208] (2) Place the homogenized sample at 30°C for 5 min to separate the nucleic acid and protein complexes.

[0209] (3) Add 200 μL of pre-cooled chloroform, cover the tube cap, oscillate for 15 s, and place at room temperature for 3 min.

[0210] (4) After ultracentrifugation at 12000 rpm at 4 ℃ for 10 min, the sample will be divided into three layers: the middle layer, the colorless aqueous ph...

Embodiment 2

[0228] Example 2: CT and PLA 2 Construction of bacmid

[0229] 1. CT and PLA2 Recovery of PCR products

[0230] Specific steps are as follows:

[0231] (1) Equilibrium column: Add 500 μL of equilibrium solution BL to the adsorption column CA2, ultracentrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the adsorption column CA2 back into the collection tube.

[0232] (2) Cut out a single target nucleic acid band, cut the cut gel into pieces, put it into a clean EP tube, place it on a balance and weigh it.

[0233] (3) Add 3 times the volume of sol solution PN to the above-mentioned EP tube. Note: The calculation method for the volume of glue is 0.1 g=0.1 mL. Then put it in a constant temperature water bath at 50 °C for 10 min, and gently turn the EP tube up and down every 2 min until the glue block is completely dissolved.

[0234] (4) Add the sol solution obtained in step 3 into the adsorption column CA2, place at 25 °C for 2 min, then ultracentrifuge at 1...

Embodiment 3

[0252] Example 3: CT and PLA 2 Construction of Insect Virus Recombinant Plasmid

[0253] 1. Competent Cell Preparation of Escherichia coli DH10Bac

[0254] (1) Spread Escherichia coli DH10Bac cells on LB (Kan+) solid plates, screen freshly activated DH10Bac single colonies, and inoculate the colonies in a 250 mL Erlenmeyer flask containing 2 mL LB liquid medium (Kan+), 37 Cultivate with shaking overnight at ℃, inoculate 0.5 mL of the overnight culture solution into a new 50 mL of the same medium the next day, and continue shaking to OD 600 The value reaches around 0.6.

[0255] (2) Under aseptic conditions, transfer the above bacterial solution to a 50 mL EP tube pre-cooled at 0°C, place on ice for 10 minutes, cool the bacterial solution to 0°C, centrifuge at 4,000 rpm for 10 minutes at 4°C, and discard the supernatant , invert the tube for several minutes to allow residual medium to flow out.

[0256] (3) Use 10 mL of 0.1 M CaCl pre-cooled at 0 ℃ 2 Resuspend the cell pe...

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Abstract

The invention discloses a method for producing cobra snake poison protein (CT) and phospholipase A2 (PLA2) in a baculovirus-insect expression system. The method comprises the following steps: separating DNA (Deoxyribonucleic Acid) sequences expressing CT and PLA2 mature peptides from the poison gland of a cobra by using a RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method, and constructing insect virus expression vectors BM-CT and BM-PLA2; transferring a vector carrying a target gene and an empty vector into Sf9 insect cells to obtain virus particles; infecting the cells by using the virus particles, detecting the expression of the target protein through SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) electrophoresis, and purifying CT and PLA2 from cell lysis supernatant by using a Ni column elution method. The analgesic activities of CT and PLA2 are detected by using the abdominal constriction test of mice. As proved by a detection result, the twisting times of the mouse in a CT treatment group are remarkably less than that of a PLA2 treatment group and a control group, indicating that the obtained CT has analgesic activity and has a better effect than PLA2; the twisting times of the mouse in the PLA2 treatment group are remarkably less than that of the control group, indicating that the obtained PLA2 also has analgesic activity. The related CT and PLA2 proteins can be taken as analgesics for application to the development and application of clinical medical analgesics as well as the research of foundation medicine.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a method for producing cobra CT and PLA in a baculovirus-insect expression system 2 Methods. Background technique [0002] There are more than 20 kinds of biologically active components in cobra venom, mainly toxic proteins, polypeptides and enzymes, etc., which are used in cancer treatment and anti-cancer, anti-tumor; hemostasis and anticoagulation; poisonous drugs and analgesics; Preparation of antivenom serum; lowering blood pressure, lowering fibrin, thrombolysis; treating blood stasis headache and other aspects have important drug activity. [0003] Neurotoxin has always been a research hotspot because of its large expression in cobra. The main medical application of neurotoxins is for analgesia and anesthesia. According to Phui Yee et al., in 2004, scientists began to use snake venom to relieve neuropathic pain, tumor pain, intractable pain and joint pain. At...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N9/16C07K1/22A61K38/17A61K38/46A61P29/00
CPCC07K14/46C12N9/18C12N15/86C12N2710/14043C12Y301/01004Y02A50/30
Inventor 蒋和生韦佩君杨秀荣鄢航潘能庆
Owner 南宁培元基因科技有限公司
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