CN115558018A - Induction method for improving expression level of antimicrobial peptide OH-CATH30 of king cobra - Google Patents
Induction method for improving expression level of antimicrobial peptide OH-CATH30 of king cobra Download PDFInfo
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Abstract
The invention provides an induction method for improving the expression level of antimicrobial peptide OH-CATH30 of king cobra, wherein the formula of a culture medium for induction expression is per 1000mL of the system, and comprises the following steps: 20g of glucose, 0.186g of calcium sulfate dihydrate, 3.6g of potassium sulfate, 2.98g of magnesium sulfate heptahydrate, 0.826g of potassium hydroxide, 5.34ml of 85% phosphoric acid, 4ml of PTM, 10g of amino acid and the balance of deionized water, wherein the amino acid is histidine, threonine and histidine combined amino acid, leucine and histidine combined amino acid or threonine and leucine combined amino acid. Inoculating pichia pastoris containing expression plasmids into the culture medium, culturing at 29 ℃ for 18 hours to enter an induction stage, culturing at 29 ℃ for 24-48 hours, centrifuging bacterial liquid, taking supernate, and filtering with a 0.22-micron filter membrane to obtain expression liquid with improved expression level. The induction method can obviously improve the expression, and the culture medium added with histidine improves the induction expression by about 2.7 percent, and the culture medium added with the amino acid composition of histidine and threonine improves the induction expression by about 80 percent.
Description
Technical Field
The invention relates to the technical field of genetic engineering biology, in particular to an induction method for improving the expression level of Oenothera carinata antimicrobial peptide OH-CATH30.
Background
cathelicidins are cationic host defense peptides that play an important role in the innate immune system, the cobra antibacterial peptide OH-CATH30 is a truncated peptide of cathelicidins, consists of 30 amino acid sequences, and is found to be applied to Escherichia coli (E. Coli) ((E. Coli))Escherichia coli) Pseudomonas aeruginosaPseudomona aeruginosa) Staphylococcus aureus (1)Staphylococcus aureus) And Enterobacter aerogenes: (A), (B)Enterobacter aerogenes) And various gram-negative bacteria and gram-positive bacteria have better bacteriostatic effects.
The experiment of induction expression of the antibacterial peptide OH-CATH30 of the king cobra prepared by genetic engineering at present shows that the recombinant pichia pastoris can secrete and express the antibacterial peptide OH-CATH30 of the king cobra in BMGY, but hardly secretes and expresses in a BSM culture medium, and the growth speed of the yeast is slow. In order to solve the problem, an induction method for improving the expression level of the antibacterial peptide OH-CATH30 of the king cobra by adding a composite component into a BSM culture medium is provided.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an induction method for improving the expression quantity of the King cobra antibacterial peptide OH-CATH30, which can improve the growth speed of recombinant pichia pastoris and increase the secretion expression of the King cobra antibacterial peptide OH-CATH30.
The purpose of the invention is realized by the following scheme:
the invention provides an induction method for improving the expression level of antimicrobial peptide OH-CATH30 of king cobra, which mainly comprises the following steps:
s01: preparing an induced expression culture medium, preparing an YPD (YPD) basal culture medium according to a conventional experimental method, and then preparing a BSM (basic fibroblast growth factor) culture medium to obtain the BSM culture medium, wherein in each 1000mL system of the BSM culture medium, the culture medium comprises the following components: 20g of glucose, 0.186g of calcium sulfate dihydrate, 3.6g of potassium sulfate, 2.98g of magnesium sulfate heptahydrate, 0.826g of potassium hydroxide, 5.34ml of 85% phosphoric acid, 4ml of PTM1, 10g of amino acid and the balance of deionized water.
S02: performing an expression experiment on the cobra antibacterial peptide OH-CATH30, inoculating pichia pastoris containing expression plasmids into a culture medium prepared in S01, culturing at 29 ℃ for 18 hours to enter an induction stage, culturing at 29 ℃ for 24 hours to 48 hours, centrifuging a bacterial liquid, extracting a supernatant, and filtering with a 0.22 mu m filter membrane to obtain an expression liquid with improved expression level.
Preferably, the amino acid in the BSM medium in the S01 step is histidine, a threonine and histidine combination amino acid, a leucine and histidine combination amino acid, or a threonine and leucine combination amino acid.
The invention has the beneficial effects that: the induction expression method can obviously improve the growth speed of the pichia pastoris, can quickly enter an induction stage, improves the induction expression amount by about 2.7 percent by adding the culture medium of histidine, improves the induction expression amount by about 80 percent by adding the culture medium of the amino acid composition of histidine and threonine, and has obvious bacteriostatic effect.
Description of the drawings:
FIG. 1: and (3) high performance liquid chromatography detection charts of 48 h-induced expression solutions of 3 types of control groups added with histidine, histidine and threonine combined amino acids.
The specific implementation mode is as follows:
the present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the concept of the invention. All falling within the scope of the invention.
The invention provides an induction method for improving the expression level of the antibacterial peptide OH-CATH30 of the king cobra, and provides a basis for large-scale fermentation of the antibacterial peptide OH-CATH30 of the king cobra.
Example 1: experiment with histidine as an amino acid
(1) Reagents and materials
The glucose is purchased from the Huffi food additive business ministry of Linyi city, orchid and mountain areas;
lysine and phenylalanine were purchased from cantonese biotechnology limited;
calcium sulfate dihydrate, potassium sulfate, tryptone, yeast extract, sodium hydroxide, ammonia water, sodium molybdate dihydrate, concentrated sulfuric acid, copper sulfate pentahydrate and boric acid are purchased from the group of Chinese medicines;
magnesium sulfate heptahydrate, potassium hydroxide, 85% phosphoric acid and iron sulfate heptahydrate are purchased from chemical reagent factories of Fochen, tianjin;
potassium iodide was purchased from west science corporation; cobalt chloride hexahydrate was purchased from Tay chemical Co Ltd in Wuxi; biotin was purchased from Biotechnology engineering (Shanghai) Inc.; zinc chloride is purchased from Tay chemical Co., ltd in Wuxi city; manganese sulfate monohydrate was purchased from Jiangsu Qiangsheng functional chemical Co., ltd;
(2) Preparation method of culture medium
(1) Preparing a YPD basal medium:
a: weighing 6g tryptone and 3g yeast extract, adding 270ml pure water for dissolving, adjusting pH to 7 with 1M sodium hydroxide solution, and autoclaving at 121 ℃ for 20min;
b: weighing 20g of glucose, adding 60ml of pure water to dissolve the glucose, fixing the volume to 100ml, and carrying out autoclaving at 108 ℃ for 30min to obtain a glucose solution with the mass concentration of 20% (W/V);
c: 30ml of a 20% (W/V) glucose solution of mass concentration was slowly added to the solution prepared in step 1 to obtain a minimal medium YPD.
(2) Preparing a BSM culture medium:
a: weighing 20g of glucose, 0.186g of calcium sulfate dihydrate, 3.6g of potassium sulfate, 2.98g of magnesium sulfate heptahydrate and 0.826g of potassium hydroxide, adding 800 mL of deionized water for dissolving, adding 5.34mL of 85% phosphoric acid, fixing the volume to 1000mL, and carrying out autoclaving at 121 ℃ for 20min;
b: weighing 3g of histidine, adding the histidine into 300mL of the solution prepared in the step (1), heating at 65 ℃ to dissolve the histidine, adjusting the pH value to 5.0 by using ammonia water, and filtering through a 0.22 mu m filter membrane for later use;
c: preparing a PTM1 solution, weighing 6g of copper sulfate pentahydrate, 0.08g of potassium iodide, 3g of manganese sulfate monohydrate, 0.2g of sodium molybdate dihydrate, 0.02g of boric acid, 0.5g of cobalt chloride hexahydrate, 20g of zinc chloride, 65g of ferric sulfate heptahydrate, 0.2g of biotin and 5mL of concentrated sulfuric acid, adding 800 mL of pure water for dissolving, and fixing the volume to 1000mL to obtain a trace salt solution PTM1;
d: and (c) adding 1.2 mL of PTM1 into the solution prepared in the step b to obtain a BSM culture medium.
(3) Expression experiment of cobra antibacterial peptide OH-CATH30
(1) Growth phase amplification of king cobra antibacterial peptide OH-CATH30
a: selecting a pichia pastoris single colony containing an expression plasmid, inoculating the pichia pastoris single colony in a 20 mLYPD culture medium, and culturing at 29 ℃ and 220rpm for 16h to prepare a primary seed solution;
b: inoculating the first-level seed solution into 100mL YPD culture medium according to the proportion of 1%, and carrying out amplification culture at 29 ℃ and 220rpm for 16h to obtain a second-level seed solution;
c: the secondary seed solution was inoculated into 300mL of the BSM medium prepared in example 2 at a ratio of 10%, and cultured at 29 ℃ and 220rpm for 66 hours. The results are shown in Table 1.
(2) Induction phase expression of king cobra antibacterial peptide OH-CATH30
a: adding methanol and sorbitol into the culture medium added with the amino acid respectively, and starting the induction expression of the recombinant yeast.
(3) Carrying out bacteriostatic experiments
a: collecting the expression solution for 48h, removing macromolecules through a 0.22 mu m filter membrane, performing a plate bacteriostasis experiment on 2 pathogenic bacteria of Staphylococcus aureus (Staphylococcus aureus) and pseudomonas aeruginosa (pseudomonas aeruginosa), culturing at 37 ℃ for about 16h, and taking out to observe the size of a bacteriostasis zone. The results are shown in Table 2.
Example 2: experiments in which the added amino acid was a combination of threonine and histidine.
The difference between this example 2 and example 1 is that the amino acid added is a threonine-histidine combined amino acid, and the component ratio is 1; the experiment was carried out by referring to the procedure of example 1, and the results are shown in tables 1 and 2.
Example 3: experiments in which the added amino acid was a combination of leucine and histidine.
The embodiment 3 is different from the embodiment 1 in that the added amino acid is a leucine and histidine combined amino acid, and the component ratio is 1; the experiment was carried out by referring to the procedure of example 1, and the results are shown in tables 1 and 2.
Example 4: experiments in which the added amino acid was a combination of threonine and leucine.
The difference between this example 4 and example 1 is that the amino acid added is a combination of threonine and leucine in a ratio of 1; the experiment was carried out by referring to the procedure of example 1, and the results are shown in tables 1 and 2.
The experimental data of the influence of the addition of 4 different amino acids on the growth rate of pichia pastoris are shown in table 1:
table 1: experimental data of influence of adding 4 different amino acids on growth speed of pichia pastoris
From the above test results, it was found that the expression level can be improved by adding histidine, a threonine and histidine combination amino acid, a leucine and histidine combination amino acid, or a threonine and leucine combination amino acid, respectively, at the growth stage, and the amounts added were all 10g/L, pH =5, 10% of inoculation, and 18h of culture at 29 ℃.
Table 2 size of zone of inhibition of 48h expression fluid
The results show that: the growth speed of the recombinant pichia pastoris can be improved in the growth stage by adding histidine, threonine and histidine combined amino acid, leucine and histidine combined amino acid or threonine and leucine combined amino acid, and the yeast can enter a rapid growth phase after being cultured for 18 hours so as to start induction. After the recombinant protein is expressed for 48 hours, taking an expression solution of the recombinant protein for carrying out an antibacterial experiment, and adding histidine or a table of threonine and histidine combined amino acids in a growth periodDaye to Staphylococcus aureus (Staphylococcus aureus) The inhibition effect is obviously improved, and the expression solution of threonine and histidine combined amino acid added in the growth period is used for treating pseudomonas aeruginosaPseudomona aeruginosa ) The inhibitory effect of (2) is significantly improved.
Example 5: detecting the expression level of the supernatant by liquid chromatography.
The expression medium containing histidine or a combination of histidine and threonine added with amino acids at the growth phase was collected for 48 hours, dialyzed and eluted, and then the expression medium was subjected to detection by reversed-phase high performance liquid chromatography, and the results are shown in fig. 1, whereby the medium containing histidine increased the induced expression by about 2.7%, and the medium containing an amino acid combination of histidine and threonine increased the induced expression by about 80%.
Claims (2)
1. An induction method for improving the expression level of the antibacterial peptide OH-CATH30 of the king cobra is characterized in that: the induction method comprises the following steps:
s01: preparing an induced expression culture medium, preparing a YPD basal culture medium according to a conventional experimental method, and then preparing a BSM culture medium to obtain the induced expression BSM culture medium; in each 1000mL system of the BSM culture medium, the culture medium comprises the following components: 20g of glucose, 0.186g of calcium sulfate dihydrate, 3.6g of potassium sulfate, 2.98g of magnesium sulfate heptahydrate, 0.826g of potassium hydroxide, 5.34ml of 85% phosphoric acid, 4ml of PTM, 10g of amino acid and the balance of deionized water;
s02: performing an expression experiment on the cobra antibacterial peptide OH-CATH30, inoculating pichia pastoris containing expression plasmids into a culture medium prepared in S01, culturing at 29 ℃ for 18 hours to enter an induction stage, culturing at 29 ℃ for 24 hours to 48 hours, centrifuging a bacterial liquid, extracting a supernatant, and filtering with a 0.22 mu m filter membrane to obtain an expression liquid with improved expression level.
2. The induction method for increasing the expression level of the antimicrobial peptide OH-CATH30 of the king cobra according to claim 1, which is characterized in that: and in the step S01, the amino acid in the BSM culture medium is histidine, a threonine and histidine combined amino acid, a leucine and histidine combined amino acid or a threonine and leucine combined amino acid.
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