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Use of CifA and CifB muteins for modulating cytoplasm incompatibility

A technology for mutating proteins and compatibility, which is applied in the field of genetic engineering of proteins to achieve the effect of solving incompatibility

Pending Publication Date: 2022-04-26
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although cytoplasmic incompatibility factors have been discovered, there are still deficiencies in the regulation of their interactions and their application in the control of mosquito-borne diseases and agricultural pests.

Method used

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  • Use of CifA and CifB muteins for modulating cytoplasm incompatibility
  • Use of CifA and CifB muteins for modulating cytoplasm incompatibility
  • Use of CifA and CifB muteins for modulating cytoplasm incompatibility

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction of pET-22b-CidAST

[0059] Using 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 as templates , with 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...

Embodiment 2

[0072] Example 2 Construction of pRS425-CidAST

[0073] 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 mutein CidAST gene whose restriction sites are BamHI and SalI.

[0074] The PCR product and the vector pRS425 were double-digested with restriction endonucleases BamHI and SalI, respectively, so that the target gene fragment and the vector had cohesive ends. The specific enzyme digestion system is shown in Table 4.

[0075] 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.

[0076] After conversion and double enzyme digestion verification, the positive results obtained were verified by sequencing. Finall...

Embodiment 3

[0077] Example 3 Construction of pRS416-CidBST

[0078] 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-ND2 Based 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 containing ...

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Abstract

The invention discloses an application of a mutant protein of artificially designed cytoplasm incompatibility factors (CI factor, Cif), including CifA (CidA, CinA, CndA) and CifB (CidA, CidB, CndB), to regulation and control of cytoplasm incompatibility, and particularly discloses a mutant protein of artificially designed cytoplasm incompatibility factors (CI factor, Cif), including CifA (CidA, CinA, CndA) and CifB (CidA, CidB, CndB). According to the CifA or CifB mutant protein disclosed by the invention, an originally compatible A factor and an originally compatible B factor are enabled to lose interaction, so that originally incompatible cytoplasm incompatible factors CifA and CifB from different Wolbachia strains are enabled to generate interaction. Experiments prove that the mutant protein disclosed by the invention has the function of a wild cytoplasmic incompatibility factor, and can eliminate the yeast growth inhibition induced by a B factor by combining the B factor so as to realize the ability of yeast rescue; and meanwhile, the yeast toxicity inducing capability of the strain is not damaged by mutation of the B factor. In addition, the mutant protein disclosed by the invention can realize the phenomenon of regulating and controlling the cytoplasm incompatibility at the fruit fly level, so that the CifA and CifB mutant proteins disclosed by the invention can be applied to regulating and controlling the cytoplasm incompatibility, and the controllable operation of the cytoplasm incompatibility is realized.

Description

technical field [0001] The invention belongs to the field of genetic engineering of proteins, and in particular relates to the application of CifA and CifB mutant proteins. 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 offspring of mosquitoes carrying different ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/29C12N15/31C12N15/70C12N15/81C12N15/85A01N37/46A01N47/44A01P7/04
CPCC07K14/29C12N15/70C12N15/81C12N15/85A01N37/46A01N47/44
Inventor 王泽方肖云杰王镐锋王炜陈侠杨海涛
Owner TIANJIN UNIV
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