Pseudosciaena crocea Cystatin recombinant protein and application thereof

Abstract

The invention provides a pseudosciaena crocea Cystatin recombinant protein and application thereof, and belongs to the technical field of gene engineering. The pichia pastoris engineering bacterium for efficiently expressing the pseudosciaena crocea Cystatin recombinant protein is constructed and is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:M 2020369. The pseudosciaena crocea Cystatin recombinant protein produced by using pichia pastoris engineering bacteria can inhibit the activity of papain, pseudosciaena crocea cathepsin B and pseudosciaena crocea cathepsin S, up-regulate the expression level of inflammation-related factors TNF alpha in macrophages, and induce the generation of an antibacterial active substance nitric oxide in the macrophages; and therefore, the pseudosciaena crocea Cystatin recombinant protein has a good application prospect as a protease inhibitor and an immunopotentiator.

Description

technical field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a large yellow croaker cysteine ​​protease inhibitor Cystatin recombinant protein and application thereof. Background technique

[0002] Cystatins are a class of natural inhibitors that bind tightly to cysteine ​​proteases and are reversible. They can regulate the function of cysteine ​​proteases, protect the body from the destructive hydrolysis of proteases from itself, bacteria or viruses, and play an important role in maintaining the homeostasis of the body, resisting bacterial and viral infections, and regulating inflammatory responses (Natasˇa Kopitar-Jerala. 2006. The role of cystatins in cells of the immune system. FEBS Letters 580: 6295–6301). According to the location, size and complexity of the polypeptide chain, members of the cysteine ​​protease inhibitor superfamily are divided into 3 types: the first type is stefins, including Cystatin A and...

Examples

Embodiment 1

[0014] Example 1 Construction of Pichia pastoris engineering bacteria that highly express recombinant protein Cystatin of large yellow croaker

[0015] The large yellow croaker Cystatin recombinant protein of the present invention is the mature peptide sequence of large yellow croaker Cystatin (amino acid 20-138, as shown in SEQ ID NO.2), and the sequence of large yellow croaker Cystatin has been published (Genbank: XM_027278065.1). Using the spleen cDNA of large yellow croaker as a template, use the primers Cystatin-F and Cystatin-R to amplify the gene fragment encoding the mature peptide of large yellow croaker Cystatin (sequence shown in SEQ ID NO.1), and then use the pEASY- Uni Seamless Cloning and Assembly Kit connects the target fragment to the yeast expression vector pPICZαA to obtain the recombinant expression vector pPICZαA-Cystatin. Recombinant vector pPICZαA-Cystatin was linearized by Mss I digestion, and transformed into Pichia pastoris SMD1168 competent cells by e...

Embodiment 2

[0018] Embodiment 2 Cystatin recombinant protein expression and purification of large yellow croaker

[0019] (1) Expression analysis of Cystatin recombinant protein in large yellow croaker

[0020] The Pichia pastoris that embodiment 1 obtains Pichia pastoris SMD1168 / pPICZαA-Cystatin engineered bacteria were inoculated into yeast medium 1, and cultured with shaking at 30 °C and 220 rpm. When the OD600 reaches 2.0, pour the above bacterial solution into a centrifuge tube, centrifuge at 6000 g at 4 °C for 7 min, remove the supernatant, resuspend the bacterial cells with yeast medium 2, and culture with shaking at 30 °C, adding 1 % methanol, induce expression for 4 days, and then use SDS-PAGE to analyze the expression of Cystatin recombinant protein in large yellow croaker in the culture supernatant. The results showed that compared with the control strain, Pichia pastoris Pichia pastoris In the culture supernatant of SMD1168 / pPICZαA-Cystatin engineering bacteria, the targe...

Embodiment 3

[0027] Inhibitory activity of embodiment 3 large yellow croaker Cystatin recombinant protein

[0028] (1) Inhibitory activity on papain

[0029]In a 100 µL reaction system, add 20 µL papain (20 nM), 10 µL 10×papain buffer, 40 µLddH 2 O, then add 10 µL of Cystatin recombinant protein from large yellow croaker with a final concentration of 0 nM, 5 nM, 10 nM, 20 nM and 25 nM, and finally mix with 20 µL of the substrate Z-FR-AMC with a final concentration of 12.5 µM , under the conditions of excitation wavelength of 380 nm and emission wavelength of 460 nm, the fluorescence released by hydrolysis of Z-FR-AMC was measured every 30 s using a multi-functional microplate reader SpectraMaxM5. The results showed that the Cystatin recombinant protein of large yellow croaker had obvious inhibitory effect on the activity of papain, and the inhibition constant was 3.012×10 -11 M( figure 2 A);

[0030] The 10×papain buffer formulation is: 4 M sodium phosphate, 40 mM EDTA, 40 mM DTT, pH ...

technical field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a large yellow croaker cysteine ​​protease inhibitor Cystatin recombinant protein and application thereof. Background technique

[0002] Cystatins are a class of natural inhibitors that bind tightly to cysteine ​​proteases and are reversible. They can regulate the function of cysteine ​​proteases, protect the body from the destructive hydrolysis of proteases from itself, bacteria or viruses, and play an important role in maintaining the homeostasis of the body, resisting bacterial and viral infections, and regulating inflammatory responses (Natasˇa Kopitar-Jerala. 2006. The role of cystatins in cells of the immune system. FEBS Letters 580: 6295–6301). According to the location, size and complexity of the polypeptide chain, members of the cysteine ​​protease inhibitor superfamily are divided into 3 types: the first type is stefins, including Cystatin A and...

Examples

Embodiment 1

[0014] Example 1 Construction of Pichia pastoris engineering bacteria that highly express recombinant protein Cystatin of large yellow croaker

[0015] The large yellow croaker Cystatin recombinant protein of the present invention is the mature peptide sequence of large yellow croaker Cystatin (amino acid 20-138, as shown in SEQ ID NO.2), and the sequence of large yellow croaker Cystatin has been published (Genbank: XM_027278065.1). Using the spleen cDNA of large yellow croaker as a template, use the primers Cystatin-F and Cystatin-R to amplify the gene fragment encoding the mature peptide of large yellow croaker Cystatin (sequence shown in SEQ ID NO.1), and then use the pEASY- Uni Seamless Cloning and Assembly Kit connects the target fragment to the yeast expression vector pPICZαA to obtain the recombinant expression vector pPICZαA-Cystatin. Recombinant vector pPICZαA-Cystatin was linearized by Mss I digestion, and transformed into Pichia pastoris SMD1168 competent cells by e...

Embodiment 2

[0018] Embodiment 2 Cystatin recombinant protein expression and purification of large yellow croaker

[0019] (1) Expression analysis of Cystatin recombinant protein in large yellow croaker

[0020] The Pichia pastoris that embodiment 1 obtains Pichia pastoris SMD1168 / pPICZαA-Cystatin engineered bacteria were inoculated into yeast medium 1, and cultured with shaking at 30 °C and 220 rpm. When the OD600 reaches 2.0, pour the above bacterial solution into a centrifuge tube, centrifuge at 6000 g at 4 °C for 7 min, remove the supernatant, resuspend the bacterial cells with yeast medium 2, and culture with shaking at 30 °C, adding 1 % methanol, induce expression for 4 days, and then use SDS-PAGE to analyze the expression of Cystatin recombinant protein in large yellow croaker in the culture supernatant. The results showed that compared with the control strain, Pichia pastoris Pichia pastoris In the culture supernatant of SMD1168 / pPICZαA-Cystatin engineering bacteria, the targe...

Embodiment 3

[0027] Inhibitory activity of embodiment 3 large yellow croaker Cystatin recombinant protein

[0028] (1) Inhibitory activity on papain

[0029]In a 100 µL reaction system, add 20 µL papain (20 nM), 10 µL 10×papain buffer, 40 µLddH 2 O, then add 10 µL of Cystatin recombinant protein from large yellow croaker with a final concentration of 0 nM, 5 nM, 10 nM, 20 nM and 25 nM, and finally mix with 20 µL of the substrate Z-FR-AMC with a final concentration of 12.5 µM , under the conditions of excitation wavelength of 380 nm and emission wavelength of 460 nm, the fluorescence released by hydrolysis of Z-FR-AMC was measured every 30 s using a multi-functional microplate reader SpectraMaxM5. The results showed that the Cystatin recombinant protein of large yellow croaker had obvious inhibitory effect on the activity of papain, and the inhibition constant was 3.012×10 -11 M( figure 2 A);

[0030] The 10×papain buffer formulation is: 4 M sodium phosphate, 40 mM EDTA, 40 mM DTT, pH ...

Images