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Expression and purification method for paraoxonase 1 gene

A paraoxonase, expression and purification technology, applied in the field of genetic engineering and protein engineering, can solve problems such as PON1 gene difficulties

Inactive Publication Date: 2015-06-24
TIANJIN YAOYU BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the difficult problem of expressing and purifying the active PON1 gene in the prior art, the present invention provides a method for expressing and purifying the paraoxonase 1 gene

Method used

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  • Expression and purification method for paraoxonase 1 gene
  • Expression and purification method for paraoxonase 1 gene
  • Expression and purification method for paraoxonase 1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Recombinant transfer vector pFastBac TM 1- PON1 build

[0023] According to the characteristics of paraoxonase 1 gene, primers F and R were designed, and cDNA was synthesized by reverse transcription using silkworm total RNA as template, and PCR amplification was carried out using cDNA as template and F and R as primers.

[0024] Upstream primer F:5 ’ -CCG GAATTC ATGGCTAAACTGACAGCG-3' (SEQ ID NO: 1), wherein the underline is Eco RI restriction site;

[0025] Downstream primer R: 5 ’_ CCG CTCGAG CTACAGCTCACAGTAAAG-3' (SEQ ID NO:2), underlined xho Ⅰ Restriction site.

[0026] The amplification procedure was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 45 s, annealing at 59°C for 30 s, extension at 72°C for 1 min, and 30 cycles; the final extension at 72°C for 8 min was recovered to obtain the paraoxonase 1 (PON1) gene.

[0027] Will PON 1 gene and pFastBac TM 1 carrier respectively with EcoR I and xho Ⅰ Carry out ...

Embodiment 2

[0028] Embodiment 2: Construction of Bombyx mori recombinant baculovirus vBm-PON1

[0029] (1) Obtaining recombinant baculovirus DNA (Bacmid-PON1)

[0030] The recombinant transfer vector pFastBac constructed in Example 1 TM 1-PON1 was transformed into Escherichia coli DH10Bac competent cells, and positive clones were obtained by blue-white screening, using four sets of primers: M13 upstream and downstream primers, PON1 upstream and downstream primers, M13 downstream primers and PON1 upstream primers, and M13 upstream primers and PON1 downstream primers Yes, carry out PCR identification, where M13F: 5′-CCCAGTCACGACGTTGTAAAACG-3′ (SEQ ID NO: 4); M13R: 5′-AGCGGATAACAATTTTCACACAGG-3′ (SEQ ID NO: 5), take a small amount of PCR products for agarose gel Electrophoresis electrophoresis, the results are as follows figure 2 As shown, it indicates that the correct Bacmid-PON1 positive clones were obtained.

[0031] (2) Recombinant baculovirus DNA (Bacmid-PON1) transfected silkworm B...

Embodiment 3

[0037] Example 3 SDS-PAGE and Western blotting analysis and identification of the fifth instar larvae inoculated with Bombyx mori recombinant baculovirus vBm-PON1

[0038] Bombyx mori recombinant baculovirus vBm-PON1 was amplified and inoculated with fifth instar larvae of Bombyx mori (Qingsong×Haoyue) by acupuncture. After the onset of the disease, the hemolymph of silkworm was collected for further experiments or stored in a -80°C refrigerator.

[0039] Treatment method for the hemolymph of silkworm larvae: filter the hemolymph through 9 layers of medical gauze, centrifuge at 2000rpm for 10min at 4°C, collect the supernatant, and resuspend the precipitate with 0.9% normal saline. SDS-PAGE and Western blotting analysis were performed on the treated silkworm hemolymph. The result is as image 3 and Figure 4 As shown, a specific band appeared near 43kD in the supernatant, indicating that the Bombyx mori recombinant baculovirus was successfully expressed in the fifth instar l...

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Abstract

Belonging to the technical field of genetic engineering, the invention discloses an expression and purification method for paraoxonase 1 gene. The method includes: firstly amplifying paraoxonase 1 gene, performing connection to a transfer vector pFastBacTM I to construct a recombinant transfer vector, transforming an Escherichia coli DH10Bac competent cell to obtain recombinant baculovirus DNA containing the paraoxonase 1 gene, transfecting a Bombyx mori BmN cell with the recombinant baculovirus DNA, carrying out replication and assembly to obtain a recombinant virus containing the paraoxonase 1 gene, then inoculating Bombyx mori fifth instar larva with the recombinant virus, taking the Bombyx mori blood after incidence, carrying out centrifugal filtration, impurity removing and concentration, then performing Cibacron Blue affinity chromatography and antibody affinity chromatography to purify the Bombyx mori blood so as to obtain paraoxonase 1 protein. The method provided by the invention realizes high-efficiency expression of protein, the structure is close to natural protein, and the problems of low content of natural PON1 and difficult purification are solved.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and protein engineering, and specifically relates to a method for expressing and purifying paraoxonase 1 gene. Background technique [0002] Paraoxonase 1 (paraoxonase1, PON1) plays an important role in the prevention and treatment of organophosphate poisoning, but it is difficult to obtain natural PON1 at present. Although PON1 expressed in Escherichia coli is cheap and has a short production cycle, the obtained protein is due to lack of Glycosylation and phosphorylation modifications have problems such as immune rejection, short half-life, and easy degradation when used in vivo. [0003] At present, although there are also reports on the expression of active PON1 gene by genetic engineering methods, the purified recombinant protein has not been obtained, and there is no relatively perfect method for the expression and purification of paraoxonase 1. Contents of the invention [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/55C12N9/16
Inventor 张耀洲谭淑敏李杰王小飞盖其静
Owner TIANJIN YAOYU BIOLOGICAL TECH
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