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Silkworm recombination baculovirus expressing paraoxonase 1 gene and preparation method and application thereof

A technology of recombinant baculovirus and paraoxonase, applied in microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as unpurified recombinant proteins, and achieve high expression, high Good security and the effect of solving insufficient sources

Inactive Publication Date: 2013-02-13
TIANJIN YAOYU BIOLOGICAL TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Ji Aiguo et al. (Ji Aiguo, Huai Yahong, Zhang Lupei, et al. Construction of bovine paraoxonase 1 gene, paraoxonase 2 and in prokaryotic expression vector and expression analysis[J]. Biotechnology Communications, 2008, 35( 9): 59-63) cloned the bovine serum paraoxonase gene, and initially expressed the active paraoxonase PON1 by genetic engineering methods, but did not obtain purified recombinant protein

Method used

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  • Silkworm recombination baculovirus expressing paraoxonase 1 gene and preparation method and application thereof
  • Silkworm recombination baculovirus expressing paraoxonase 1 gene and preparation method and application thereof
  • Silkworm recombination baculovirus expressing paraoxonase 1 gene and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Synthesis of PON1 gene

[0045] According to the PON1 gene sequence published by GenBank accession number AY499193.1, a pair of primers were designed to amplify the PON1 gene (italics are restriction sites), and its nucleotide sequence is shown in SEQ ID NO:1.

[0046] Upstream primer F: 5'-TAT GAATTC ATGGCTAAACTGACAGCG-3’, where italics are Eco RI site;

[0047] Downstream primer R: 5'-GCG CTCGAG TTACAGCTCACAGTAAAG-3', where italics are xho I site;

[0048] Using the pBlueScript-PON1 plasmid (Teng Xia, Yang Shen, etc. Expression of human serum Q-type paraoxonase in Escherichia coli. [J] Biotechnology Communications 2007, 18 (1): 015-018) as a template, using the above Primers were used for PCR amplification and agarose gel electrophoresis (such as figure 1 As shown), the size of the gene fragment of PON1 is about 1065bp, and agarose gel electrophoresis shows that the size of the PCR product of the target gene in corridor 2 is consistent with the ac...

Embodiment 2

[0059] Example 2: Recombinant transfer vector pFastBac TM Construction of HTA-PON1

[0060] PON1 gene and pFastBac obtained in embodiment 1 TM For HTA vector (Invitrogen) EcoR I and xho Ⅰ (Fermentas Company) performed double enzyme digestion at the same time, and agarose gel electrophoresis was used to recover the digested product; the recovered PON1 gene fragment was connected to the digested pFastBac with Ligation high ligase (Fermentas Company) TM On the HTA vector, make the target gene (PON1 gene) under the control of the polyhedron promoter, transform Escherichia coli TG1 competent cells (Fermentas company), screen positive clones, and obtain the recombinant transfer vector pFastBac TM HTA-PON1. The recombinant transfer vector was carried out by PCR and double enzyme digestion, and the product was identified by agarose gel electrophoresis (such as figure 2 shown), two bright bands appear in the double enzyme digestion product of No. 1 lane, one of which is the 106...

Embodiment 3

[0061] Embodiment 3: Construction of Bombyx mori recombinant baculovirus BmNPV-PON1

[0062] 1) Obtaining recombinant baculovirus DNA (Bacmid-PON1)

[0063] The recombinant transfer vector pFastBac constructed in Example 2 TM HTA-PON1 was transformed into Escherichia coli DH10Bac competent cells (Fermentas company), and positive clones were obtained by blue-white screening. PCR was performed using the combination of M13 universal primers and the upstream and downstream primers of the target gene, and a small amount of PCR products were taken for agarose gel electrophoresis. (Such as image 3 Shown), electrophoresis results showed that the correct Bacmid-PON1 positive clones were obtained.

[0064] 2) Recombinant baculovirus DNA (Bacmid-PON1) transfected silkworm BmN cells

[0065] a. Take 1ml of silkworm BmN cells in good growth state with a pipette 8-12 hours in advance and spread them in a small cell culture dish (3.5cm in size);

[0066] b. Take 10 μL of recombinant Bac...

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Abstract

The invention relates to silkworm recombination baculovirus expressing a paraoxonase 1 gene and a preparation method and application thereof and belongs to the technical field of biopharmacy. By means of correlation technique of gene engineering, the paraoxonase 1 gene is built in a silkworm baculovirus expressing system, and then a silkworm bioreactor is used for expressing target protein. The silkworm bioreactor is used for expressing the paraoxonase 1 gene and conducting activity detection of the paraoxonase 1 gene, efficient expression of the protein is achieved, modification such as glycosylation is increased compared with the target protein expressed through prokaryotic expression, and foundation for further researching functions of the target protein is provided.

Description

technical field [0001] The invention relates to a silkworm recombinant baculovirus expressing paraoxonase 1 gene, a preparation method and application thereof, and belongs to the technical field of biopharmaceuticals. Background technique [0002] Paraoxonase 1 (paraoxonase1, PON1) widely exists in a variety of microorganisms, insects, snake venoms, squid axons, squid posterior salivary glands, birds and mammals, and is a broad-spectrum non-specific esterase. It can degrade organophosphoric acid, aromatic hydroxy acid ester and carbamate by catalyzing the hydrolysis of phosphate bonds, thus playing an important role in hydrolyzing organophosphorus pesticides and degrading nerve agents. Therefore, paraoxonase 1 is expected to be a new approach for the prevention and treatment of organophosphate poisoning. According to reports, PON1 not only plays an important role in the detoxification of organophosphorus poisons, but also is closely related to the pathogenesis of atheroscl...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/55C12N15/63C12N9/16A61K38/46A61P9/10A61P3/10A61P31/04A61P1/16C12R1/93
Inventor 张耀洲李霞李晓瑞陈剑清舒特俊
Owner TIANJIN YAOYU BIOLOGICAL TECH
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