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Method for purifying cryptosporidium protein kinase

A purification method and technology of Cryptosporidium, applied in the field of in vitro recombinant protein purification, can solve the problems of pure protein, unguaranteed activity, hinder systematic research on the invasion mechanism of Cryptosporidium, etc., and achieve the effect of improving solubility

Pending Publication Date: 2019-05-10
EAST CHINA UNIV OF SCI & TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Because cryptosporidium protein kinases often appear in fragmented expression or self-cleavage degradation during the expression process in vitro, it is difficult to obtain relatively pure protein through conventional purification methods, and the activity cannot be guaranteed, which affects subsequent protein functions and enzymes. The scientific research also hinders the systematic study of the invasion mechanism of Cryptosporidium

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  • Method for purifying cryptosporidium protein kinase
  • Method for purifying cryptosporidium protein kinase
  • Method for purifying cryptosporidium protein kinase

Examples

Experimental program
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Embodiment 1

[0056] A method for purifying Cryptosporidium calcium-dependent protein kinase 1 (CDPK1) protein, comprising the following steps:

[0057] 1. Construction of CpCDPK1 protein recombinant expression system

[0058] Using Cryptosporidium DNA as a template, specific amplification primers (SEQ ID NO: 1-2) were designed, and the CpCDPK1 gene fragment (SEQ ID NO: 3) was obtained by PCR amplification. The electrophoresis of the amplified product is as follows: figure 1 shown.

[0059] The amplified CpCDPK1 gene fragment and the pET-28a plasmid were digested with EcoRI and XhoI, and the digested product was purified and ligated with T4 ligase to obtain the CpCDPK1 recombinant plasmid, which was then plated and single clones were picked. 1. Confirm the correctness of the recombinant plasmid after PCR sequencing verification, and name it pET28a-CpCDPK1.

[0060] 2. Plasmid transduction and recombinant protein expression

[0061] Transfer the pET28a-CpCDPK1 plasmid into the expression...

Embodiment 2

[0086] A method for purifying Cryptosporidium calcium-dependent protein kinase 9 (CDPK9) protein, comprising the following steps:

[0087] 1. Construction of CpCDPK9 protein recombinant expression system

[0088] Using Cryptosporidium DNA as a template, the CpCDPK9 gene fragment (SEQ ID NO: 6) was amplified by PCR with specific primers (SEQ ID NO: 4-5). The electrophoresis of the amplified product is as follows: Image 6 shown.

[0089] The amplified CpCDPK9 gene fragment and the pET-28a plasmid were digested with EcoRI and XhoI, and the digested product was purified and ligated with T4 ligase to obtain the CpCDPK9 recombinant plasmid, which was then plated and single clones were picked. 1. Confirm the correctness of the recombinant plasmid after PCR sequencing verification, and name it pET28a-CpCDPK9.

[0090] 2. Plasmid transduction and recombinant protein expression

[0091] Transfer the pET28a-CpCDPK9 plasmid into the expression strain BL21, culture it by streaking, pic...

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Abstract

The invention discloses a method for purifying cryptosporidium protein kinase. Firstly, an in-vitro recombination expression vector of cryptosporidium protein kinase is constructed; then plasmid transduction and IPTG low-temperature inducible expression are carried out; after successfully expressed thallus is split, protein is purified through rubber tapping recycling. According to the method forpurifying cryptosporidium protein kinase, by means of low-temperature inducing and IPTG concentration improving, the solubility of expression protein is improved, the problem that during in-vitro expression of cryptosporidium protein kinase, inclusion body expression is likely to autotomizing degradation is solved through a rubber tapping recycling method, high-purity single-strip recombinant protein with enzyme activity can be obtained, and the purifying effect on protein hard to express and large in molecular weight can be improved. The purified protein can be further used for functional analysis and studies, and a foundation is laid for solving various problems in the field of cryptosporidium protein functions.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method for purifying an in vitro recombinant protein of Cryptosporidium protein kinase. Background technique [0002] Cryptosporidium ( Cryptosporidium ) is a widely distributed and serious pathogen of diarrheal diseases. Cryptosporidium is a parasitic protozoan of the Apicomplexan phylum, including more than 70 known species and genotypes. A disease that infects almost all vertebrates and causes diarrhea, especially persistent watery diarrhea in young animals. The infection rate of Cryptosporidium to young cattle, sheep and pigs is very high (>30%), and the cumulative infection rate to livestock within one year old is 100%. It is also the main pathogen of dairy cow diarrhea in many countries. Therefore, this disease causes serious harm to aquaculture. [0003] In addition, Cryptosporidium is also an important zoonotic pathogen, and it is the second pathogen ...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/70C12N1/21
CPCY02A50/30
Inventor 冯耀宇苏嘉缘张强李娜郭亚琼
Owner EAST CHINA UNIV OF SCI & TECH
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