CN115820773A - Culture medium formula for induction expression of weever antibacterial peptide wb-molonecidin - Google Patents

Abstract

The invention provides a culture medium formula for induction expression of weever antibacterial peptide wb-molonecidin, wherein in each 1L system of the culture medium, the culture medium comprises the following components: 20g of glucose, 0.465g of calcium sulfate dihydrate, 9g of potassium sulfate, 7.45g of magnesium sulfate heptahydrate, 2.065g of potassium hydroxide, 13.35mL of 85% phosphoric acid, 1mL of PTM, 10g of amino acid and the balance of deionized water, wherein the amino acid is lysine, phenylalanine or a combination amino acid of lysine and phenylalanine, the culture medium formula disclosed by the invention can ensure the good growth of the recombinant Pichia pastoris, and the recombinant Pichia pastoris can enter a logarithmic phase after growing for 18 hours, so that the inducible expression can be carried out; meanwhile, the expression level of the recombinant protein and the expression quantity of the recombinant protein are obviously improved in the induction stage.

Description

Culture medium formula for induction expression of weever antibacterial peptide wb-molonecidin

Technical Field

The invention relates to the technical field of genetic engineering biology, in particular to a culture medium formula for induced expression of weever antibacterial peptide wb-morronicidin.

Background

The weever antibacterial peptide wb-molonecidin consists of 28 amino acid sequences, has broad-spectrum antibacterial activity and is found to be resistant to aeromonas veronii (Aeromonas veronii) ((R))Aeromonas veronii) Listeria monocytogenes (L.), (Listeria monocytogenes) The two gram-negative bacteria and gram-positive bacteria have good bacteriostatic effects.

A culture medium commonly used in the process of expressing the foreign protein by the pichia pastoris is a BSM universal culture medium, but the BSM universal culture medium has the advantage that the effect of only using the universal BSM culture medium for expression is different because the components cannot completely meet the growth requirement of the pichia pastoris containing target protein gene plasmids. The applicant finds that in a gene engineering expression and preparation experiment of the weever antibacterial peptide wb-molonecidin, when the pichia pastoris containing the weever antibacterial peptide wb-molonecidin plasmid is cultured at high density by taking BSM as a universal basal medium, the expression level of the recombinant protein is lower and the biological activity is also lower. Therefore, the induction expression level and the biological activity of the weever antibacterial peptide wb-molonecidin are needed to be improved by optimizing the components of the culture medium.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a culture medium formula for induction expression of weever antibacterial peptide wb-morronicidin.

The purpose of the invention is realized by the following scheme:

a culture medium formula for induction expression of weever antimicrobial peptide wb-molonecidin is characterized in that: the formula of the culture medium is that amino acid is added on the basis of BSM culture medium, aiming at improving the induced expression level and the biological activity of the weever antibacterial peptide wb-morronicidin.

The culture medium comprises the following components in percentage by weight:

1L of the medium was prepared, and added:

20g of glucose;

0.465g of calcium sulfate dihydrate;

9g of potassium sulfate;

magnesium sulfate heptahydrate 7.45g;

2.065g of potassium hydroxide;

13.35mL of 85% phosphoric acid;

PTM1 4mL；

amino acid 10g

Dissolving the culture medium components with deionized water, and fixing the volume to 1L.

Preferably, the amino acid is lysine, phenylalanine or a combination of lysine and phenylalanine.

The positive progress effects of the culture medium formula for the induction expression of the weever antibacterial peptide wb-molonecidin provided by the invention are as follows: the culture medium formula can ensure that the recombinant pichia pastoris grows well, and the recombinant pichia pastoris enters a logarithmic phase after growing for 18 hours, so that induced expression can be carried out; meanwhile, the expression level of the recombinant protein and the expression quantity of the recombinant protein are obviously improved in the induction stage. The culture medium provided by the invention obviously improves the bacteriostatic activity of the weever antibacterial peptide wb-morronicidin.

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 it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.

The invention provides a culture medium formula for induction expression of weever antimicrobial peptide wb-morronicidin, and provides a foundation for large-scale fermentation of the weever antimicrobial peptide wb-morronicidin.

Example 1: preparation method of culture medium

(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 cantor biotechnology limited, guanzhou;

calcium sulfate dihydrate, sulfuric acid, copper sulfate pentahydrate, boric acid, tryptone, yeast extract, sodium hydroxide, potassium sulfate, manganese sulfate, sodium molybdate dihydrate, ammonia water, sodium chloride, disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate 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;

NB liquid medium refers to nutrient broth medium, purchased from Hangzhou microbial agents, inc.;

(2) Preparation of the culture Medium

(1) Preparing a YPD basal culture medium:

a: weighing 6g of tryptone and 3g of yeast extract, adding 270mL of pure water for dissolving, adjusting the pH value to 6.0 by using 1M of sodium hydroxide solution, and then sterilizing at 121 ℃ for 20min under high temperature and high pressure;

b: weighing 20g of glucose, adding 60mL of pure water to dissolve the glucose, fixing the volume to 100mL, and sterilizing the glucose for 30min at 108 ℃ under high temperature and high pressure to obtain a glucose solution with the mass concentration of 20% (W/V);

c: 30mL of a 20% (W/V) glucose solution of step b was slowly added to the solution prepared in step a to obtain a minimal medium YPD.

(2) Preparing a BSM culture medium:

a: weighing 20g of glucose, 0.465g of calcium sulfate dihydrate, 9g of potassium sulfate, 7.45g of magnesium sulfate heptahydrate and 2.065g of potassium hydroxide, adding 800 mL of deionized water for dissolution, adding 13.35mL of 85% phosphoric acid, fixing the volume to 1000 mL, and carrying out autoclaving at 121 ℃ for 20min;

b: weighing 3g of the lysine, the phenylalanine or the combined amino acid of the lysine and the phenylalanine which are matched according to the proportion of 1, adding the weighed combined amino acid into 300mL of the solution prepared in the step (1), heating the solution at 65 ℃ to dissolve the combined amino acid, adjusting the pH value to 5.0 by using ammonia water, and filtering the solution by using 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, 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 1000 mL to obtain a trace salt solution PTM1;

d: and (c) adding 11.2 mL of PTM into the solution prepared in the step (b) to obtain the BSM culture medium.

Example 2: expression experiment of weever antibacterial peptide wb-morronicidin

(1) Growth phase amplification of pichia pastoris containing weever antibacterial peptide wb-morronicidin recombinant expression plasmid

a: selecting a pichia pastoris single colony containing an expression plasmid, inoculating the single colony in a 20 mLYPD culture medium, and culturing at 29 ℃ and 220rpm for 16h;

b: inoculating the overnight-cultured bacterial liquid into 100mL YPD medium according to the proportion of 1%, and carrying out amplification culture at 29 ℃ and 220rpm for 16h;

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 influence of the specific addition of 3 amino acids on the growth rate of pichia pastoris is shown in table 1:

table 1: experimental data of influence of adding 3 different amino acids on growth speed of pichia pastoris

From the above experimental results, it was found that the growth rate of yeast can be increased by adding lysine at the growth stage, the addition amount is 10g/L, pH =5.0, inoculating 10%, and culturing at 29 ℃ for 18 h.

(2) Induction phase expression of pichia pastoris containing weever antimicrobial peptide wb-moronecidin recombinant expression plasmid

a: adding methanol and sorbitol into the 3 kinds of culture medium added with different amino acids, respectively, and performing induced expression on the recombinant yeast.

(3) Collection and dialysis of expression fluid

a: weighing 2.922g of sodium chloride; 16.69g disodium hydrogen phosphate dodecahydrate; adding ultrapure water into 0.5034g of sodium dihydrogen phosphate, and diluting to a constant volume of 1L to obtain dialysis buffer solution;

b: collecting the expression solution of 3 different amino acids and a control group in the step (1) for 48h, removing insoluble large-particle substances in the expression solution through a 0.22-micron filter membrane, dialyzing for 12h in a buffer solution at the temperature of 4 ℃, dialyzing for 12h with ultrapure water, and collecting the expression solution after dialysis;

c: freeze-drying the expression solution after dialysis, and dissolving with sterile water to obtain a weever antibacterial peptide sample, wherein the concentration is defined as 0.1 mg/mL.

(4) Carrying out bacteriostatic experiments

a: the experimental strain was inoculated into NB liquid medium, cultured at 37 ℃ and 180rpm until the logarithmic growth phase, and the OD was measured ₆₀₀ Value, dilution of the broth to 4X 10 with fresh NB broth ⁵ cfu/mL。

Adding 0.5mL of weever antibacterial peptide sample into 0.5mL of culture medium to serve as a 1 st tube, uniformly mixing, adding 0.5mL of weever antibacterial peptide sample into a 2 nd tube, sequentially diluting in multiple times, sucking 0.5mL out from a 4 th tube, discarding, and taking the 5 th tube as a control tube as shown in Table 2:

TABLE 2 dilution method

Adding 0.1mL of diluted weever antibacterial peptide sample into a sterile 96-well plate added with 0.1mL of bacterial liquid, repeating each sample for 3 times, uniformly mixing, standing at 37 ℃ for overnight culture for about 12h, and measuring light absorption at 600nm by using an enzyme-linked immunosorbent assay. The minimum inhibitory concentration is the lowest concentration of the sample at which bacterial growth is not visible to the naked eye. The results are shown in Table 3.

TABLE 3 Perch antimicrobial peptide sample Minimum Inhibitory Concentration (MIC)

The results show that: the growth speed of the recombinant pichia pastoris can be improved in the growth stage by adding lysine, phenylalanine or the combination of the 2 amino acids, the recombinant pichia pastoris can enter the logarithmic growth phase after being cultured for 18 hours, the state of the recombinant strain is good, but benzene is addedThe effect of alanine was not significant. MIC experiment results show that expression liquid of recombinant pichia pastoris added with lysine and phenylalanine combined amino acid in growth period is used for treating pseudomonas aeruginosaP aeruginosa) Staphylococcus aureus (1)S. aureus) Aeromonas hydrophila (b) ((b))A hydrophila) Aeromonas veronii (A), (B) and (C)A veronii) And Listeria monocytogenes (L.), (L monocytogene) The minimum bacteriostatic concentration of the recombinant protein is obviously reduced relative to a control group, which shows that the recombinant protein expression quantity can be improved by adding the lysine and phenylalanine combined amino acid, and the bacteriostatic effect is also obviously improved. While lysine or phenylalanine alone can only enhance the effect on Staphylococcus aureus: (S. aureus) The inhibitory effect of (3).

Claims (1)

1. A culture medium formula for induction expression of weever antimicrobial peptide wb-molonecidin is disclosed, wherein the culture medium comprises the following components in percentage by weight:

1L of the medium was prepared, and added:

20g of glucose;

0.465g of calcium sulfate dihydrate;

9g of potassium sulfate;

magnesium sulfate heptahydrate 7.45g;

2.065g of potassium hydroxide;

13.35mL of 85% phosphoric acid;

PTM1 4mL；

10g of amino acid;

dissolving the components of the culture medium by using deionized water, and fixing the volume to the final volume of 1L, wherein the amino acid is lysine, phenylalanine or the combination of lysine and phenylalanine.