Expression vector of nuclease protein Cas9 and construction method and expression purification of expression vector of nuclease protein Cas9

A technology of nuclease protein and expression vector, which is applied in the field of expression vector construction, can solve the problems of high cost, high market price of Cas9 protein, and complicated operation, and achieve the effects of low cost, promotion of popularization and development, and simple operation

Pending Publication Date: 2019-11-26
NANJING UNIV OF SCI & TECH
View PDF3 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the presence of rare codons in the transcription and translation of nucleic acid sequences, this expression requires a specific host bacterium; and a large amount of culture medium is required to obtain the protein Cas9, which is costly; and three systematic purification steps are required to ...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Expression vector of nuclease protein Cas9 and construction method and expression purification of expression vector of nuclease protein Cas9
  • Expression vector of nuclease protein Cas9 and construction method and expression purification of expression vector of nuclease protein Cas9
  • Expression vector of nuclease protein Cas9 and construction method and expression purification of expression vector of nuclease protein Cas9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The gene cloning of embodiment 1 nuclease Cas9

[0041] 1. Cloning of Thioredoxin Trx cDNA

[0042] a) Design a pair of primers according to the pET32a plasmid purchased from MERK Company. The oligonucleotide sequence upstream of the primer is shown in SEQ ID No.1, and the oligonucleotide sequence downstream of the primer is shown in SEQ ID No.2. Using the pET32a plasmid as a template, the cDNA of thioredoxin-histidine tag-tobacco etch virus protease recognition site was obtained through PCR reaction, the sequence of which is shown in SEQ ID No.7. The PCR reaction system configuration is shown in Table 1.

[0043] Table 1 PCR reaction system preparation table

[0044]

[0045]

[0046] PCR results such as figure 1 shown. The first lane is Marker, the second lane is the cDNA of the target fragment △Trx-His, and the length of the target fragment is 528bp.

[0047] b) Recover the cDNA of the thioredoxin-histidine tag-tobacco etch virus protease recognition site, ...

Embodiment 2

[0056] The expression mode of embodiment 2 nuclease Cas9

[0057] a) Induced expression of fusion protein Trx-His-Cas9

[0058] The correctly identified pET32a-△Trx-His-Cas9 plasmid was transferred into Escherichia coli expression host Tuner (DE3) to obtain a stable transformed single colony; the single colony was inoculated into the LB liquid medium containing 100 μg / ml ampicillin, at 200 rpm at 37°C overnight to obtain overnight bacteria. Inoculate the overnight bacteria into 750mL LB medium, the inoculation ratio is 1:250, and cultivate to OD at 230 rpm, 30°C 600 After reaching 0.8-1.0, add IPTG (isopropylthiogalactopyranoside) with a final concentration of 0.5 mM, and then culture at 25° C. for 10 hours. The cells were collected by centrifugation at 4000 rpm for 15 minutes. The bacteria were resuspended in PBS buffer, and then centrifuged at 4000 rpm for 15 minutes to obtain the Trx-His-Cas9 bacteria containing the fusion expression protein Trx-His-Cas9 of thioredoxin-n...

Embodiment 3

[0063] Example 3 Activity detection of nuclease Cas9 protein

[0064] a) Psy1-sgRNA

[0065] Search the NCBI database to obtain the gene sequence of phytoene synthase psy, and design sgRNA according to the mutation site in "White Mutants of Chlamydomonas reinhardtii Are Defective in Phytoene Synthase". A pair of primers were designed according to the sgRNA to introduce the T7 promoter and the targeting sequence. The oligonucleotide sequence upstream of the primer is shown in SEQ ID No.5, and the oligonucleotide sequence downstream of the primer is shown in SEQ ID No.6. Using the PCR product of sgRNA as a template, use T7 RNA polymerase to transcribe Psy1-sgRNA as shown in Table 2. After denaturation at 65°C for 10 minutes, run the gel directly after cooling on ice. The results are as follows: Figure 7 shown. After phenol / chloroform extraction, the purified Psy1-sgRNA was obtained, and 600ng samples were taken to run on the gel, and the results were as follows Figure 8 sho...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses an expression vector of a nuclease protein Cas9 and a construction method and expression purification of the expression vector of the nuclease protein Cas9. According to the expression vector of the nuclease protein Cas9, an original thioredoxin sequence in pET32a is replaced with cDNA of an amplified thioredoxin-histidine-tag-tobacco-etch-virus-protease recognition site, and codons are optimized to a Cas9 gene sequence which is easy to express in a host and has nuclear localization signal peptide and 3xFLAG tag sequence fused in a C-terminal to be integrated into the thioredox in sequence. According to the expression vector of the nuclease protein Cas9 and the construction method and expression purification of the expression vector of the nuclease protein Cas9, codon optimization and thioredoxin fusion expression are used for obtaining a large amount of the nuclease Cas9 protein, under the condition that only an LB medium is used, 10 mg of a highly purified active Cas9 protein can be finally obtained per gram of escherichia coli Tuner (DE3) wet bacteria, and high expression of the high-fidelity nuclease protein Cas9 at a low cost is realized; and the requirements on the host are not high, and the obtained protein can be used for large-scale biological in vitro assembly and in vivo gene editing experiments.

Description

technical field [0001] The invention belongs to the technical field of expression vector construction, and relates to the construction of a nuclease protein expression vector, in particular to an expression vector of the nuclease protein Cas9, a construction method and expression purification thereof. Background technique [0002] CRISPR (Clustered regularly interspaced short palindromic repeats) is called regularly clustered interspaced short palindromic repeats, derived from bacteria, is a system for bacteria to protect themselves against viruses, and can be widely used to delete, add, activate or inhibit various organisms target gene. Thus, CRISPR-Cas9 is a precise and versatile genetic weapon. [0003] According to "High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wideoff-target effects", four amino acid mutations of Streptococcus pyogenes Cas9 (SpCas9) can reduce the off-target effect of its gene editing, but the mutation sites are N497 The protease activ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N9/22C12N15/62C12N15/63
CPCC12N9/22C12N15/63C07K2319/09C07K2319/43C07K2319/35C12N2800/22
Inventor 周敏陈沐王德孚卢颖洪王坤
Owner NANJING UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap