Visual tracking method for rapid expression of NS1 protein by bombyx mori bioreactor

A bioreactor, silkworm technology, applied in the field of genetic engineering, can solve the problems of low expression, difficult protein expression, low efficiency, etc.

Inactive Publication Date: 2013-12-11
JIANGSU UNIV
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  • Abstract
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Problems solved by technology

[0005] However, the genome of Bm-Bacmid is relatively large, and it is replicated in E. coli DH10B cells in a low-copy form. Secondly, the efficiency of liposome-mediated recombinant Bm-Bacmid transfection in BmN cells is relatively low. At the same time, research It shows that there are some proteins with special physical and chemical properties that are often difficult to express or have low expression levels in BEVS. Therefore, it is a good idea to quickly identify whether recombinant virions are produced in cells transfected with recombinant Bm...

Method used

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  • Visual tracking method for rapid expression of NS1 protein by bombyx mori bioreactor
  • Visual tracking method for rapid expression of NS1 protein by bombyx mori bioreactor
  • Visual tracking method for rapid expression of NS1 protein by bombyx mori bioreactor

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Embodiment 1

[0023] Embodiment 1. recombinant plasmid build

[0024] In order to amplify the promoter of the very early gene ie1 in the baculovirus BmNPV genome, two specific primers IE1-F-:5'- were designed by Primer Premier5.0 software TACGTA GTAAGTTATTAATAAAATGCACTGACACG-3' (SnaBI) (SEQ ID NO. 1) and IE1-R:5'- GGATCC ATCGTGTCGCCGCCA-3'(BamH I) (SEQ ID NO.2), amplified a 560bp DNA fragment from the BmNPV genome by PCR, carried out SnaBI and BamH I single enzyme digestion to the amplified DNA fragment respectively, and digested The target DNA was gel-cut and purified, and the purified DNA was cloned into the pFastHTB vector to generate the recombinant plasmid pFastHTB-P ie1 ; For amplifying the full length of the egfp gene sequence, two specific primers EGFP-F:5'- were designed by Primer Premier5.0 software GGATCC ATGGTGAGCAAGGGC-3'(BamHI) (SEQ ID NO.3) and EGFP-R:5'- GGTACC TTACTTGTACAGCTCGTCCATG-3'(KpnI)(SEQ ID NO.4), a 720bp full-length sequence of the egfp gene was amplified...

Embodiment 2

[0027] Embodiment 2. recombinant plasmid build

[0028] In order to reveal the relationship between the phosphorylation modification of NS1 protein of Bombyx mori didensovirus and its function, the silkworm bioreactor was used to express NS1 protein on a large scale. Design two specific primers ns1-F:5'-AT by Primer Premier5.0 software GCTAGC ATGGAATCGAAGTCAAATTT-3'(Nhe I) (SEQ ID NO.9) and ns1-R:5'-TA CTCGAG CTACCCATAATATTTATTATACG-3'(xho I) (SEQ ID NO.10), using the BmBDV viral genome as a template, specifically amplifies the ns1 gene from the pMD18T-ns1 plasmid by PCR. Carry out Nhe I / xho I double enzyme digestion on the amplified target DNA fragment, and recombine the recovered enzyme digestion product with the plasmid constructed above connect, generate The recombinant plasmid was identified by PCR with different primer pairs, and the results were as follows figure 2 As shown, it indicates that the recombinant donor plasmid has been successfully generated and ...

Embodiment 3

[0029] Embodiment 3. recombinant bacmid Identification

[0030] Take 5ng of the constructed recombinant plasmid Add Escherichia coli DH10B competent cells, mix gently, place on ice for 30min, heat shock at 42°C for 45sec, place on ice for 5min, add 900μl of SOC medium, shake vigorously (225rpm) at 37°C for 4h, take 100μl of suspension Liquid coating on the three antibody containing K + T + G + (50 μg / ml kanamycin, 10 μg / ml tetracycline, 7 μg / ml gentamicin) and 40 μg / ml IPTG and 20 mg / ml X-gal LB plate, cultivated at 37 ° C for 36 to 48 hours, and selected typical comparative The large white single colonies were cultured, and the extracted Bacmid was identified by multiple rounds of PCR through different primer pairs, and the results were as follows image 3 shown.

[0031] In the above reaction: kanamycin, tetracycline and gentamycin were purchased from Sigma Company, and IPTG and X-gal were purchased from Shanghai Chaorui Biological Company.

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Abstract

The invention relates to a visual tracking method for rapid expression of an NS1 protein by a bombyx mori bioreactor. The method comprises the steps: a donor plasmid carrying an egfp expression cassette and an nsl gene expression cassette is constructed, an scherichia coli DH10B competent cell is converted by the donor plasmid, the egfp expression cassette and the nsl gene expression cassette which are between Tn7R-Tn7L of sequences of the donor plasmid can be specifically transposed onto a Bm-Bacmid vector, an integrated recombinant eukaryotic expression vector for expressing an egfp gene and an nsl gene is constructed, a bombyx mori is infected by recombinant buddedvirus BV particles produced after insect cells are transfected with the integrated recombinant eukaryotic expression vector, and real-time monitoring of a situation of virus multiplication in a bombyx mori body is carried out through visual green fluorescent signals. The method can perform large-scale and real-time monitoring of expression dynamic situation of an exogenous gene in the bombyx mori body, and lays the foundation for revealing characteristics of the NS1 protein and other functional proteins.

Description

technical field [0001] The invention relates to genetic engineering technology, and discloses a construction process and application of a silkworm bioreactor visualization tracking expression system. Specifically, it refers to a preparation method of recombinant budding virion BV, and the use of this virus to rapidly and efficiently express the non-structural protein NS1 of the silkworm bipartite densovirus in the silkworm bioreactor, which is the subsequent function and function of the NS1 protein. This method is also applicable to the expression of other foreign genes and recombinant protein drugs with important functions. Background technique [0002] The baculovirus-insect cell expression system (BEVS) is one of the four major expression systems for genetic engineering today. Most of the baculovirus expression vectors used are the A. californica nuclear polyhedrosis virus (AcNPV) or the silkworm nuclear polyhedrosis virus. body virus (BmNPV). Among many expression syst...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/66
Inventor 李国辉王鹏唐琦胡朝阳姚勤陈克平李芒芒徐五
Owner JIANGSU UNIV
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