Vector and method for eukaryotic soluble expression of polar-tube glycoprotein of Nosema bombycis

A technology of silkworm microparticles and glycoproteins, which is applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of glycosylation modification characteristics of natural polar tube proteins and the inability to glycosylation modification , Difficulty in extracting and purifying polar tube protein, etc., to achieve the effect of simple separation and purification technology

Inactive Publication Date: 2018-11-30
SOUTHWEST UNIV
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  • Abstract
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Problems solved by technology

[0003] The polar tube protein NbPTP1 of Bombyx mori is only soluble in reducing agents such as DTT and β-mercaptoethanol, and it is extremely difficult to extract and purify the polar tube protein with glycosylation characteristics.
Bombyx mori microsomum polar duct protein heterologously expressed by the E. coli prokaryotic expression system does not have the characteristics of natural polar duct protein glycosylation modification, so it is impossible to study the function of its glycosylation modification

Method used

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  • Vector and method for eukaryotic soluble expression of polar-tube glycoprotein of Nosema bombycis
  • Vector and method for eukaryotic soluble expression of polar-tube glycoprotein of Nosema bombycis
  • Vector and method for eukaryotic soluble expression of polar-tube glycoprotein of Nosema bombycis

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Embodiment 1

[0024] Embodiment 1, pSLI / V5-His expression plasmid vector construction containing IE2 promoter

[0025] According to the OpIE2 and the multiple cloning site region sequence (1-947bp) on the pIZ / V5-His vector, a pair of primers with AscI restriction sites were designed, and the pIZ / V5-His vector DNA was used as a template for PCR amplification. The upstream primer is: 5'-ggcgcgccggatcatgatgataaacaat-3' (SEQ ID NO.1), the downstream primer is: 5'-ggcgcgcctgttctttcctgcgttatcc-3' (SEQ ID NO.2), and the amplification condition is 94°C pre-denaturation for 5 minutes; 94 Denaturation at °C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1 minute, a total of 30 cycles; finally extension at 72°C for 10 minutes, and storage at 4°C to obtain the insert fragment OpIE2-MCS. The OpIE2-MCS fragment and the intermediate vector pSLfa1180fa were respectively digested with the restriction endonuclease AscI, transformed into Escherichia coli after ligation, positive clone...

Embodiment 2

[0026] Embodiment 2, the amplification of NbPTP1 gene of Bombyx mori microspore

[0027]The PCR amplification of the polar tube glycoprotein NbPTP1 gene of Bombyx mori was based on the genome DNA of Bombyx mori, and the genome DNA of Bombyx mori was extracted by CTAB method. According to the genome sequence of M. mori, combined with NCBI sequence alignment, Primer 5.0 software was used to design primers, and an appropriate restriction site was selected in the multiple cloning site of the pSLI / V5-His expression vector. The upstream primer contained a BamHI restriction site : 5'-cgcggatccatgagaattagatccttcaaa-3' (SEQ ID NO.3), the downstream primer contains an XhoI restriction site: 5'-ccgctcgaggcgtttagcacacatggattatt-3' (SEQ ID NO.4), the amplification condition is 94°C pre-denaturation 4 Minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 90 seconds, a total of 30 cycles; last extension at 72°C for 10 minutes, and storage at 4°...

Embodiment 3

[0029] Embodiment 3, construction of the recombination vector of the Bombyx mori microspore pole tube glycoprotein NbPTP1 gene containing the Metallothionein promoter

[0030] The present invention uses pMT / Bip / V5-His A to transfect the vector, and adopts a two-step cloning method to carry out vector construction: the PCR product of NbPTP1 gene is cut and recovered, connected with the vector pMD19-T vector, transformed into Escherichia coli, and screened positive Cloning, after sequencing verification, it shows that the cloned fragment is the target fragment, and there is no amino acid mutation, and the construction of the subcloning vector can be carried out in the next step. The pMD19-NbPTP1 recombinant plasmid was digested with BglII and XhoI respectively, and the NbPTP1 gene was recovered; the pMT / Bip / V5-His A plasmid was digested with BglII and XhoI, and the pMT / Bip / V5-His A vector fragment was recovered, and the recovered NbPTP1 The gene was connected with the pMT / Bip / V5...

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Abstract

The invention relates to a vector and method for eukaryotic soluble expression of polar-tube glycoprotein of Nosema bombycis. The method comprises the following concrete steps: constructing a recombinant vector containing the NbPTP1 gene of Nosema bombycis, and then transfecting the sf9 cell of Spodoptera frugiperda for expression, wherein the expression of the recombinant vector containing the NbPTP1 gene of Nosema bombycis is regulated by an IE2 promoter. The method of the invention successfully realizes the soluble expression of the polar-tube glycoprotein of Nosema bombycis, and has a glycosylation modification, so a theoretical foundation is laid for further understanding of the functions of the polar-tube protein of Nosema bombycis and explication of the infection mechanism of Nosemabombycis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a eukaryotic carrier for soluble expression of the microsomum glycoprotein of the silkworm, and a method for soluble expression of the glycoprotein of the microsomia of the silkworm. Background technique [0002] Microsporidium silkworm is a typical species of the genus Microsporidia. Its rapid horizontal transmission and vertical transmission through eggs cause the silkworm microparticle disease, which has caused great harm to the sericulture industry in my country and even the world. Microsporidia have a special infection structure - pole tube. Polar tubes are mainly composed of three proteins: PTP1, PTP2 and PTP3. The study found that PTP1, the most abundant protein on the polar canal, is an O-mannosylated protein. It is speculated that the protein modified by glycosylation can effectively inhibit the degradation of the polar canal in the host body, and at the same time benefit PTP...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/30C07K14/44
CPCC07K14/44C12N15/85
Inventor 龙梦娴周泽扬李春峰谭瑶瑶于滨
Owner SOUTHWEST UNIV
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