Nosema bombycis hypothetical protein NB29, recombinant expression vector containing nosema bombycis hypothetical protein NB29 and application of recombinant expression vector

A technology of silkworm microparticles and hypothetical proteins, applied in applications, vectors, nucleic acid vectors, etc., can solve the problems of inability to accurately determine virus-carrying individuals, time-consuming workload, and high technical requirements for operators

Active Publication Date: 2019-03-29
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, different methods have different advantages and disadvantages. For example, microscopic examination of female moths has high technical requirements for operators, and it is impossible to fully analyze the poisoning of offspring; drug treatment can only be processed on a large scale, and it is impossible to accurately determine the presence of viruses. Toxic individuals; PCR and other molecular biology detection operations are relatively simple, but often time-consuming and heavy workload

Method used

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  • Nosema bombycis hypothetical protein NB29, recombinant expression vector containing nosema bombycis hypothetical protein NB29 and application of recombinant expression vector
  • Nosema bombycis hypothetical protein NB29, recombinant expression vector containing nosema bombycis hypothetical protein NB29 and application of recombinant expression vector
  • Nosema bombycis hypothetical protein NB29, recombinant expression vector containing nosema bombycis hypothetical protein NB29 and application of recombinant expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Bioinformatics analysis and secretion verification of silkworm microparticles NB29

[0038] The nucleotide sequence of the silkworm microparticle hypothetical protein gene NB29 was obtained, and its sequence is shown in SEQ ID No.1. Use the corresponding website to predict nuclear localization signals, signal peptides and transmembrane domain analysis.

[0039] According to bioinformatics analysis, NB29 contains a signal peptide sequence with two nuclear localization signals, such as figure 1 shown. NB29, Ps87, and Mg87 signal peptide sequences were designed, the sequences of which are shown in SEQ ID No.2, SEQ ID No.3, and SEQ ID No.4, respectively, and then sent to Gensript to synthesize the sequences.

[0040]The synthetic signal peptide sequence was cleaved by Xba I and Kpn I restriction sites at the same time, and then ligated to the yeast signal sequence trap vector pSUC2T7M13ORI that had undergone the same digestion, and the ligated product was trans...

Embodiment 2

[0042] Example 2, identification of secreted protein NB29 self-interaction

[0043] From Example 1, it was verified that the silkworm microparticle NB29 is a secreted protein, which meets the requirement of being secreted into host cells. In order to further construct the system, NB29 is first truncated according to the structural domain, and the truncated NB29-A (full length), NB29-B, NB29-C, and NB29-D sequences are designed, and the sequences are as SEQ ID No.1, SEQ ID No.5, SEQ ID No.6, and SEQ ID No.7 are shown, and then sent to Gensript Company to synthesize the sequence. NB29-A, NB29-B, NB29-C and NB29-D utilize Kpn I and BamH I to connect to the pIZ-DsRed carrier (the DsRed fluorescent protein sequence as shown in SEQ ID No.9 is connected to Hind III and Kpn of the pIZ carrier 1 restriction site), the ligation product was transformed into Escherichia coli DH5α competent cells, then positive clones were screened with an LB plate containing bleomycin (Zeocin), a single ...

Embodiment 3

[0047] Example 3, Screening of secreted protein NB29 optimal truncation

[0048] On the basis of the self-interaction of the secreted protein NB29-B, the truncated NB29-N3 (SEQ ID NO.8) was further screened out and sent to Gensript to synthesize the sequence, in order to lay the foundation for the fusion of Cas9. First, pIZ-NB29-N3 was transfected into BmN-SWU1 cells, the method was similar to Example 2, the difference was that after blocking with the blocking solution, the cells were lightly washed with 1×PBST for 3 times, each time for 5 min; the mouse Myc antibody was incubated at room temperature for 1 h, and then washed with Wash cells lightly with 1×PBST 3 times, 5 min each time; add Alexa555-labeled goat anti-mouse secondary antibody IgG and nuclear dye DAPI, treat at 37°C in the dark for 1 hour, wash cells 3 times with 1×PBST lightly, 5 min each time . Take out the cover slip and observe its location in the cells by fluorescence, the results are as follows: Figure 8...

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Abstract

The invention discloses nosema bombycis hypothetical protein NB29, a recombinant expression vector containing the nosema bombycis hypothetical protein NB29 and an application of the recombinant expression vector. A nucleotide sequence of the nosema bombycis hypothetical protein NB29 is shown in SEQ ID NO.1, expressed protein is secretory, a truncation form N3 is screened out according to the nosema bombycis hypothetical protein NB29, fusion Cas9 protein is stably expressed in silkworm cytoplasm, during infection of nosema bombycis, secretory protein NB29-B and NB29-N3 of nosema bombycis interact, Cas9 is dragged in a host cell nucleus, and the purpose of host gene editing is achieved. The Cas9 medicated inducible gene editing vector can stably edit host genes, a new idea is provided for preparing insect nosema bombycis death causing models and nosema bombycis control, reference is provided for control of nosema bombycis and other pathogens capable of infecting humans, and meanwhile, afoundation is laid for functional research of related genes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the hypothetical protein NB29 of silkworm microparticles, and also relates to the truncated fragment of the secreted protein NB29 and its recombinant expression vector and application. Background technique [0002] The silkworm is a model insect of the order Lepidoptera and has important economic value. Silkworm microparticles can cause a devastating blow to silkworms through horizontal and vertical transmission, and are the only detection objects in the silkworm egg quarantine process. At present, the most commonly used methods for the prevention and control of silkworm microparticles are microscopic examination of female moths, drug treatment, and molecular biological detection such as PCR, and finally burning seed treatment. However, different methods have different advantages and disadvantages. For example, microscopic examination of female moths has high technical requirements ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/30C07K14/44C12N15/85C12N15/90
CPCC07K14/44C12N15/85C12N15/902C12N2800/105C12N2810/10
Inventor 潘敏慧鲁成周亮董战旗陈鹏杨基贵
Owner SOUTHWEST UNIVERSITY
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