Silkworm PABP (polyadenylic acid bonding protein) bonding protein interacting factor gene BmPaip1 as well as recombinant expression vector and application thereof

A technology that combines proteins and expression vectors, applied in applications, genetic engineering, plant gene improvement, etc., can solve the problems of high transcription level and low translation efficiency of exogenous genes, so as to promote the practical process, improve translation efficiency, and improve expression volume effect

Active Publication Date: 2014-05-07
SOUTHWEST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In view of this, the purpose of the present invention is to provide a silkworm PABP-binding protein interaction factor gene BmPaip1, which solves the problem of high exogenous gene transcription level but low translation efficiency in the transgenic silkworm in the prior art; the second purpose of the present invention is to Provide a recombinant expression vector containing the silkworm PABP binding protein interaction factor gene BmPaip1, which can efficiently express the BmPaip1 gene; the third purpose of the present invention is to provide the application of the silkworm PABP binding protein interaction factor gene BmPaip1

Method used

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  • Silkworm PABP (polyadenylic acid bonding protein) bonding protein interacting factor gene BmPaip1 as well as recombinant expression vector and application thereof
  • Silkworm PABP (polyadenylic acid bonding protein) bonding protein interacting factor gene BmPaip1 as well as recombinant expression vector and application thereof
  • Silkworm PABP (polyadenylic acid bonding protein) bonding protein interacting factor gene BmPaip1 as well as recombinant expression vector and application thereof

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Embodiment 1

[0020] Embodiment 1, the molecular cloning of silkworm BmPaip1 gene

[0021]The human Paip1 gene (Homo sapiens poly(A) binding protein interacting protein 1, PAIP1, GI: 208022642) sequence downloaded from NCBI was homologously compared in silkworm SilkDB, and a homologous sequence BGIBMGA009981-TA was retrieved. The sequence was spliced ​​by EST and found to have a complete ORF. According to the comparison results, the primers for the coding region of silkworm BmPaip1 were designed: the upstream primer was BmPaip1-F: 5'-aaaa agatct atgaataacggtgatattagtgct-3' (SEQ ID NO.1), the underline is the restriction endonuclease site BglⅡ; the downstream primer is: BmPaip1-R: 5'-aaaa gcggccgc ttatcgctttatctgattcgatat-3' (SEQ ID NO.2), the underline is the restriction endonuclease site NotI. PCR amplification was carried out using the silkworm large silkworm cDNA as a template. The amplification conditions were: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 sec...

Embodiment 2

[0022] Example 2. Functional detection of the silkworm BmPaip1 gene

[0023] Cut BmPaip1 from the pMD18-T vector with BglII and NotI restriction enzymes, and connect it to the psl1180[hr3BmAct4-114LUCSer1PA] backbone vector (also known as pSL[hA4LUCSer1PA]) digested by BamHI and NotI to form a recombinant Vector pSL[hA4BmPaip1Ser1PA], such as figure 2 Shown in A (the vector psl1180[hr3BmAct4-114LUCSer1PA] is recorded in the Chinese patent publication No. 102492692A). Depend on figure 2 A shows that the recombinant vector contains an Hr3 enhancer, which can increase the expression of downstream genes, and the silkworm actin A4 gene promoter drives the expression of the silkworm BmPaip1 gene.

[0024] In order to detect whether the BmPaip1 gene has the function of promoting the expression level of the transgenic gene in the insect body, the expression vector pSL[hA4LUCSer1PA] containing the luciferase reporter gene LUC ( figure 2 B) Mix with pSL[hA4BmPaip1Ser1PA] express...

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Abstract

The invention discloses a silkworm PABP (polyadenylic acid bonding protein) bonding protein interacting factor gene BmPaip1. The nucleotide sequence of the gene BmPaip1 is shown as SEQIDNO.3, the open reading frame of the gene BmPaip1 is 1194bp long, composed of eight exons and codes 398 amino acids, theoretical protein molecular weight is 44kDa, and isoelectric point pI is equal to 5.02; and SMART online software analysis shows that the bits from 100 to 398 in the amino acid sequence of the gene BmPaip1 of a silkworm are highly homologous with an eIF4G protein of an eucaryon, and expression detection in insect cells shows that expression level of a reporter gene LUC (luciferase) can be obviously improved in cells genetically modified by the gene BmPaip1 of the silkworm, so that the gene BmPaip1 of the silkworm can be combined with eIF4E and eIF4A to form a translation initiation complex and can be used for improving translation efficiency of an exogenous gene.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the silkworm PABP-binding protein interaction factor gene BmPaip1, and also relates to a recombinant expression vector containing the gene and an application of the gene. Background technique [0002] With the rapid development of bioengineering and genetic engineering in the 21st century, people's demand for functional proteins for various purposes such as medical, medicinal, edible, beauty, and health care is increasing, and protein extraction from natural sources cannot meet the rapid growth. market demand. Establishing and improving various high-efficiency prokaryotic and eukaryotic expression systems, and using strains, cells, and insects as host bioreactors is an effective and sustainable way to achieve low-cost, large-scale production of recombinant foreign proteins with biological activity. method, and has become a research hotspot in the world today. Using Chinese hamster ov...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/63C12N15/85
Inventor 夏庆友王峰徐汉福袁林王元成赵萍向仲怀
Owner SOUTHWEST UNIV
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