A codon-optimized Chlamydia trachomatis ctl0286 gene and its application

A Chlamydia trachomatis, codon optimization technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem of protein expression failure

A Chlamydia trachomatis, codon optimization technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem of protein expression failure

CN113373163BActive Publication Date: 2022-06-03NANTONG UNIVERSITY
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  • A codon-optimized Chlamydia trachomatis ctl0286 gene and its application
  • A codon-optimized Chlamydia trachomatis ctl0286 gene and its application
  • A codon-optimized Chlamydia trachomatis ctl0286 gene and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The codon-optimized ct10286 gene sequence is shown in SEQ ID NO.1.

[0034]

[0036]

[0038]

[0039] The PCR product was added with 1 μL of DpnI, digested at 37°C for 1 h to remove the template plasmid, and stored at -20°C.

[0041]

[0042] 6. The recombinant product is transformed. The above recombinant products were immediately transformed into Escherichia coli DH5α competent cells and spread on the phase

[0049] (7) Invert overnight culture in a 37°C incubator.

[0054] 9. Identification of protein expression. Figure 2 is Coomassie brilliant blue staining to verify the expression of CTL0286 protein. Take the above 1mL culture

[0056] 11. Protein purification. The above-purified inclusions were resuspended in 10 mL of denatured lysate (25 mM HEPES

Embodiment 2

[0058] Preparation of CTL0286 polyclonal antibody. Replace the NT-CTL0286 protein solution obtained above with PBS buffer

[0059] Figure 3 shows the ability of the CTL0286 polyclonal antibody to recognize the protein. Figure A shows the identification of antibodies from 4 different mice

[0060] The function of the purified protein was verified by protein binding experiments. Figure 4 shows the binding energy of CTL0286 to cRNAPβ' subunit

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Abstract

The invention discloses a codon-optimized Chlamydia trachomatis ctl0286 The gene and its application belong to the technical field of biomedicine. The codon-optimized Chlamydia trachomatis ctl0286 Gene, the sequence of which is shown in SEQ ID NO.1. The present invention realizes the in vitro expression of the Chlamydia trachomatis protein CTL0286 that cannot be expressed in the E. coli system through a codon optimization strategy, and the antibody prepared by using the expressed protein can be used for the identification of the CTL0286 protein; as a specific protein of unknown function in Chlamydia trachomatis, the present invention It can provide the basis for functional research and antibody development of CTL0286 protein, and then provide new targets and drugs for clinical treatment and identification of chlamydia.

Description

A codon-optimized Chlamydia trachomatis ctl0286 gene and its application technical field The invention belongs to the technical field of biomedicine, be specifically related to a kind of codon-optimized Chlamydia trachomatis ct10286 Genes and their applications. Background technique As we all know, gene transcription is inseparable from the participation of RNA polymerase, chlamydia RNA polymerase (cRNAP) core enzyme Homologues of the α, β, β′ subunits of α, β and β′ have been found, but until now, whether the ω factor exists in Chlamydia has a similar function things have not yet been confirmed. The determination of chlamydia promoter in vitro transcriptional activity often borrows Escherichia coli RNA polymerase (eRNAP) to simulate cRNAP, the inventors speculate that if there is a functional analog of ω factor in Chlamydia, then it is related to the ω factor of eRNAP. The homology should be relatively high. The inventor starts from the ω protein sequence of E...

Claims

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Application Information

Patent Timeline
03 Jun 2022
Publication
CN113373163B
IPC
C12N15/31; C12N15/70; C07K14/295; C07K16/12; C07K16/06; G01N33/569; A61K38/16; A61P27/02; C12R1/19
CPC
C07K14/295; C12N15/70; C07K16/125; C07K16/06; G01N33/56927; A61K38/164; A61P27/02; C12N2800/22
Inventors
包小峰; 陆春风