Preparation and application of cryptosporidium protein kinase 660 C-terminal protein

Abstract

The present invention discloses a preparation method and an application of cryptosporidium protein kinase 660 C-terminal protein. The preparation method comprises the following steps: S1, using amplification primers shown in SEQ ID NO:1-2 to conduct PCR amplification reaction to obtain a Cp 660 C gene segment; S2, carrying out double enzyme digestion on the Cp 660 C gene fragment and a prokaryoticexpression plasmid and conducting connection to obtain a Cp 660 C recombinant plasmid; S3, transferring the Cp 660 C recombinant plasmid into an expression host bacterium and picking a positive clonebacterium; S4, carrying out amplification culture on the positive clone bacterium until a growth logarithmic phase and carrying out induced expression; S5, carrying out refrigerated centrifugation onbacterial liquid after the induced expression, collecting bacterium body, discarding supernatant, washing the bacterium body, carrying out re-suspension, adding a protease inhibitor, carrying out icebath ultrasonic lysis on the bacterium body to obtain a lysate, then carrying out low-temperature centrifugation to obtain supernatant, and conducting filtering with a filter membrane; and S6, takingthe filtered supernatant in the step 5, purifying the supernatant through a Ni column, eluting the supernatant, and collecting a target protein. The method can obtain the recombinant protein with high purity and high antigen specificity, and the purified protein can be further used for analyzing and researching functionality of the cryptosporidium protein.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically, relates to the preparation and application of an in vitro recombinant protein of Cryptosporidium protein kinase 660 C-terminal protein. Background technique [0002] Cryptosporidium is a zoonotic parasitic protozoan that is prevalent all over the world and has a wide range of hosts, including wild animals, companion animals or economic animals, and even humans can be infected with Cryptosporidium. Cryptosporidiosis mainly causes mild to severe diarrhea and vomiting, especially in young individuals, causing anorexia, watery diarrhea, abdominal pain, vomiting, fatigue, and low-grade fever. The course of cryptosporidiosis depends on the immunity of the host body. Self-limited diarrhea and persistent gastrointestinal diseases occur in a well-immune body, which becomes a host of worms; Such as infants, it will cause severe diarrhea, and even cause death. [0003] Cryptosporidium c...

AI Technical Summary

Problems solved by technology

Because the molecular weight of this kind of protease is too large, and its function has not been determined, there are often problems such as micro-expression or even failure to express or self-cleavage degradation during the expression process in vitro, which affects the subsequent research on protein function and enzymology

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