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Cytoplasm incompatibility factor CifA or CifB mutant gene and protein

A technique for mutating proteins and mutating genes, applied in genetic engineering, plant genetic improvement, recombinant DNA technology, etc.

Pending Publication Date: 2022-04-19
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no report of mutating the A and B factors from different strains, so as to realize the interaction between the A and B factors from different strains, and at the same time achieve CI rescue at the level of yeast and Drosophila

Method used

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  • Cytoplasm incompatibility factor CifA or CifB mutant gene and protein
  • Cytoplasm incompatibility factor CifA or CifB mutant gene and protein
  • Cytoplasm incompatibility factor CifA or CifB mutant gene and protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Construction of embodiment 1 pET-22b-CidAST

[0099] The wMel CidA nucleotide sequence shown in SEQ ID NO.9 in Drosophila melanogaster and the wPip CidA nucleotide sequence shown in SEQ ID NO.10 in Culex pipiens are: Template to wPip CidA-CidB ND1-ND2 (The nucleotide sequence of wPip CidB is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12) and the structure of wMel CidA, wherein the A and B factor interaction interfaces can be divided into three, the The amino acid residues involved in the interaction between CidA and CidB in wPip CidA were replaced on wMel CidA, and the CidAST sequence was designed, as shown in SEQ ID NO.1. Using the above artificially synthesized SEQ ID NO.1 as a template, using F1 as a forward primer, and R1 as a reverse primer, the gene sequence containing CidAST obtained by PCR was digested and ligated with T4 DNA ligase to mutate The protein CidAST gene was constructed on the PET-22b vector to obtain the PET-22b-CidAS...

Embodiment 2

[0112] Example 2 Construction of pRS425-CidAST

[0113] The vector was selected as pRS425, the restriction sites were BamHI and SalI, primers were designed for PCR, and the primers were named F2 and R2 respectively, and the PCR system was shown in Table 3. Using the pET-22b-CidAST constructed in Example 1 above as a template, PCR can obtain the second sequence containing the mutant protein CidAST gene whose restriction sites are BamHI and SalI.

[0114] The PCR product and the vector pRS425 were double-digested with restriction endonucleases BamHI and SalI, respectively, so that sticky ends appeared on the target gene fragment and the vector. The specific enzyme digestion system is shown in Table 4.

[0115] The target gene fragment with cohesive ends and the vector pRS425 were ligated using T4 DNA ligase, and the ligation system was shown in Table 5 above.

[0116] After conversion and double enzyme digestion verification, the positive results obtained were verified by seque...

Embodiment 3

[0117] Construction of embodiment 3 pRS416-CidBST

[0118] The wMel CidB nucleotide sequence shown in SEQ ID NO.17 in Drosophila melanogaster and the wPip CidB nucleotide sequence shown in SEQ ID NO.11 from Culex pipiens are: Template to wPip CidA-CidB ND1-ND2Based on the structure of wPip CidB, the amino acid residues involved in the interaction between CidA and CidB in wPip CidB were replaced on wMel CidB, and the CidBST sequence was designed, as shown in SEQ ID NO.3. Using the above-mentioned artificially synthesized SEQ ID NO.3 as a template, F3 as a forward primer, and R3 as a reverse primer, the gene sequence containing CidBST obtained by PCR was digested and ligated with T4 DNA ligase to mutate The protein CidBST gene was constructed on the PET-22b vector to obtain the PET-22b-CidBST plasmid; the PET-22b-CidBST plasmid was used as a template, F4 was used as a forward primer, and R4 was used as a reverse primer to perform PCR to obtain the second The sequence containin...

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Abstract

The invention discloses a mutant gene and protein of cytoplasm incompatibility factors (CI factor, Cif) comprising CifA (CidA, CinA and CndA) and CifB (CidA, CidB and CndB), the parent CifA and CifB proteins are derived from Wolbachia, the mutant protein has a plurality of amino acid mutations relative to the parent, and the nucleotide sequences of the mutant protein are as follows: CidAST is shown as SEQ ID NO.1, CidBST is shown as SEQ ID NO.3, CinAST is shown as SEQ ID NO.5, and CinBST is shown as SEQ ID NO.7. The invention also discloses a method for preparing the mutant gene. On the basis of a compound structure of cytoplasm incompatibility factors CifA and CifB, according to interaction interface characteristics of a factor A and a factor B, interaction interface mutation is carried out on an original CifA or CifB gene by utilizing a genetic engineering method, so that the originally compatible factor A and factor B lose the interaction capability, and the CifA or CifB gene is obtained. The present invention relates to a method for producing cytoplasmic incompatibility factors, CifA and CifB, from different strains of Wolbachia, which are capable of interacting with each other.

Description

technical field [0001] The invention belongs to the field of protein genetic engineering, and in particular relates to a mutant gene of cytoplasmic incompatibility factors CifA and CifB and a preparation method. Background technique [0002] Mosquito-borne diseases have always endangered human life and safety. Millions of people are infected with dengue fever, chikungunya fever and Zika virus every year, which has become a global public health security problem. As an important vector organism, mosquitoes are the focus of the control of mosquito-borne infectious diseases. With the development of biological mosquito control technology, the control of mosquito vectors and mosquito-borne diseases based on insect symbiotic bacteria Wolbachia has gradually become a research and application hotspot. Wolbachia is capable of causing cytoplasmic incompatibility (CI), whereby Wolbachia-infected males mate with uninfected females, resulting in nonviable offspring; moreover, mating offs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/31C12N15/66C12N15/81C12N1/19C12R1/865
CPCC07K14/195C12N15/66C12N15/81
Inventor 王泽方肖云杰王镐锋王炜陈侠杨海涛
Owner TIANJIN UNIV
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