CN114196695A - Construction method of high-activity traditional Chinese medicine feed additive trypsin - Google Patents

Abstract

The invention discloses a construction method of trypsin as a high-activity traditional Chinese medicine feed additive, belonging to the technical field of genetic engineering. The invention discloses a construction method of trypsin as a high-activity traditional Chinese medicine feed additive, which obtains the trypsin with higher activity at 37 ℃ by gene mutation technology (error-prone PCR), and the specific enzyme activity is 2436(BAEE unit)/mg. The enzyme has wide application prospect in the field of feed and has important significance for the breeding industry.

Description

Construction method of high-activity traditional Chinese medicine feed additive trypsin

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a construction method of trypsin which is a high-activity traditional Chinese medicine feed additive.

Background

Trypsin is used more and more widely in feed additives due to its efficacy in promoting nutrient absorption and healing of intestinal wounds. Trypsin is an active enzyme that breaks down human proteins into amino acids, which are then absorbed and utilized by the human body. The trypsin plays an important biological role in the digestion process of food, has the functions of digestive enzyme and limiting decomposition, can promote thick sputum and blood clots to be decomposed and diluted, and is convenient for a human body to discharge. In addition, the trypsin has the biological effect of resisting inflammation, promotes the proliferation of granulation tissues of wounds and can accelerate the healing of wound surfaces.

The trypsin can promote nutrient absorption, so that in the feeding of pigs, chickens, ducks, fishes and the like, one of the components of the traditional Chinese medicine feed additive is added into the feed together with the traditional Chinese medicines, and the traditional Chinese medicines have synergistic effect, so that the trypsin has good effects of promoting nutrient absorption, rapidly fattening and the like.

The activity of the existing trypsin is low in the intestinal environment, particularly in the environment of 37 ℃. The development of trypsin with higher activity at 37 ℃ has important significance for the breeding industry.

Therefore, the problem to be solved by the technical personnel in the field is to provide a construction method of the trypsin which is a high-activity traditional Chinese medicine feed additive.

Disclosure of Invention

In view of the above, the invention provides a construction method of trypsin which is a high-activity traditional Chinese medicine feed additive.

In order to achieve the purpose, the invention adopts the following technical scheme:

a construction method of a high-activity traditional Chinese medicine feed additive trypsin comprises the following specific steps:

(1) carrying out error-prone PCR by using SEQ ID NO.2 as a template and PF and PR as primers;

the primer sequences are as follows:

PF：5’-cggaattcATGACCCAATACCATCGTCGT-3’；SEQ ID NO.3；EcoR I；

PR：5’-ccaagcttTTAGTGGTGGTGGTGGTGGT-3’；SEQ ID NO.4；Hind III；

the error-prone PCR reaction system is as follows: error-prone PCR Mix 3.0 μ L, error-prone PCR dNTP 3.0 μ L, 5mM MnCl₂3.0 μ L, 10 μ g/μ L DNA template 1 μ L, 10 μ M PF 1 μ L, 10 μ M PR1 μ L, Taq DNA polymerase 1 μ L, sterilized double distilled water 17 μ L;

the error-prone PCR reaction procedure was as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 deg.C for 1min, annealing at 45 deg.C for 1min, extension at 72 deg.C for 4min, and 32 cycles; extending for 10min at 72 ℃;

(2) carrying out agarose gel electrophoresis and gel recovery on the error-prone PCR product;

(3) and carrying out double enzyme digestion, connection and transformation on the recovered error-prone PCR product and pET-28a (+) to E.coli BL21(DE3) competent cells, and carrying out expression and purification to obtain the high-activity trypsin, wherein the amino acid sequence of the trypsin is shown in SEQ ID No. 6.

Further, step (3) uses Ni-NTA purification medium to purify the protein.

According to the technical scheme, compared with the prior art, the invention discloses a construction method of trypsin which is a high-activity traditional Chinese medicine feed additive, and the trypsin with higher activity at 37 ℃ is obtained by a gene mutation technology (error-prone PCR), and the specific enzyme activity is 2436(BAEE unit)/mg. The enzyme has wide application prospect in the field of feed and has important significance for the breeding industry.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a drawing showing a map of pET28a-Typ vector of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Coli BL21(DE3) strain was purchased from beijing baiolabockik technologies ltd; plasmid pET-28a (+) was purchased from a fenghui organism.

Agarose, tris hydrochloride, ethylene diamine tetraacetic acid, nucleic acid dye 4S Green, glycine, sodium dodecyl sulfate, bromophenol blue, glycerol, dithiothreitol, Coomassie brilliant blue G250, absolute ethanol, glacial acetic acid, acrylamide, N, N-methylenebisacrylamide, ammonium persulfate, N, N-tetramethylethylenediamine, sucrose, glycerol, isopropyl-beta-D-thiogalactopyranoside, and imidazole are all purchased from Biotechnology engineering (Shanghai) GmbH; kanamycin was purchased from saiko biotechnology limited liability company; yeast extract, peptone, chromatographic grade methanol, protein Marker were purchased from Saimer Feishell science; nucleic acid Marker was purchased from Anolon (Beijing) Biotechnology Ltd; biphenyl, p-hydroxybiphenyl, was purchased from the Aladdin group. The general chemical reagents sodium chloride and calcium chloride are purchased from national drug group limited company. benzoyl-L-arginine ethyl ester (BAEE) was purchased from Shanghai drum minister Biotechnology, Inc.

The 2xTaq Plus PCRmastermix kit, the plasmid miniprep kit and the gel recovery kit are purchased from Tiangen Biotechnology (Beijing) Ltd.

EcoR I and HindIII restriction enzymes, T4 DNA ligase, were purchased from NEB.

The Ni-NTA purification media was purchased from Biotechnology engineering (Shanghai) Inc.

Example 1

1) Synthesis of Trypsin Gene

Trypsin (GenBank: ROT73769.1) of Antarctic prawn is used as an initial sequence, and the amino acid sequence of the Trypsin is shown as SEQ ID NO. 1.

mtqyhrrarlppvspraddggmrllvlfllfmvevcvqsavapshlqhllssrivggedavqgefpfqvsiehvgilgrnhicggivlsatdvltaahcisrfsehnidivaginrlsdtssnrqtveavhfiihedfnditmgsdiaiirlkhalelnewvqpvvmpktgetaqegemctvvgwgatfedgdlsdvlqkvsvpiqsdsycrsaygysavedsmlcagspeggadacdgdrggpmlcrghlhgisswgegcghsfypgvytevsaflhwnpkstedleleallgddsaapeetpsddpevqvvvaespedhdvprelppev；SEQ ID NO.1。

The trypsin codon is optimized according to the preference of the escherichia coli, and sent to the Kinsley company for synthesizing genes, the synthesized base sequence is shown as SEQ ID NO.2, and the synthesized base sequence is provided with restriction enzyme cutting sites and histidine tags.

gaattcATGACCCAATACCATCGTCGTGCACGTCTGCCACCTGTATCTCCTCGTGCAGATGATGGTGGTATGCGTCTGCTGGTGCTGTTCCTGCTGTTTATGGTTGAAGTCTGTGTTCAGTCTGCTGTAGCTCCGTCTCACCTGCAGCATCTGCTGTCTTCCCGTATCGTTGGTGGTGAAGACGCTGTTCAGGGCGAGTTCCCTTTCCAGGTGAGCATCGAACATGTTGGTATTCTGGGCCGTAACCATATCTGCGGCGGCATTGTGCTGAGCGCGACCGATGTACTGACTGCAGCGCACTGTATCTCTCGCTTCAGCGAACACAACATCGACATTGTTGCCGGTATCAACCGTCTGAGCGACACCTCTTCCAACCGTCAGACCGTGGAAGCAGTTCACTTCATCATCCACGAAGATTTCAACGACATTACTATGGGTAGCGACATTGCAATTATCCGCCTGAAACACGCGCTGGAACTGAACGAATGGGTCCAGCCGGTCGTTATGCCGAAAACCGGCGAAACCGCACAAGAAGGTGAAATGTGTACTGTAGTTGGCTGGGGTGCGACCTTCGAGGATGGTGATCTGTCCGACGTTCTGCAGAAAGTTTCCGTTCCAATTCAGTCTGATAGCTACTGCCGTTCTGCCTACGGTTACTCTGCGGTCGAAGATTCTATGCTGTGTGCAGGCTCTCCTGAAGGTGGTGCTGATGCCTGTGATGGTGATCGTGGTGGCCCGATGCTGTGTCGTGGTCACCTGCACGGTATCTCTTCTTGGGGTGAAGGCTGCGGTCATAGCTTTTATCCGGGTGTCTATACTGAGGTAAGCGCTTTCCTGCACTGGAACCCGAAAAGCACCGAAGACCTGGAGCTGGAAGCTCTGCTGGGTGATGACTCTGCTGCACCGGAGGAAACCCCGTCTGATGACCCGGAAGTACAGGTGGTTGTTGCGGAATCTCCGGAAGACCATGATGTTCCGCGCGAACTGCCGCCGGAAGTGCACCACCACCACCACCACTAAaagctt；SEQ ID NO.2。

2) Amplification of target Gene

Taking a synthesized gene sequence SEQ ID NO.2 as a template, designing a PCR amplification primer by using Snapgen software assistance, wherein the synthesis of the primer is completed by Shanghai's work, and the specific primer sequence is as follows:

an upstream primer PF: 5' -cggaattcATGACCCAATACCATCGTCGT-3'; SEQ ID No. 3; the underlined part is an EcoR I enzyme digestion site;

a downstream primer PR: 5' -ccaagcttTTAGTGGTGGTGGTGGTGGT-3'; SEQ ID No. 4; the underlined sections are HindIII sites.

The target gene was amplified using the synthetic genome as a template using 2xTaq Plus PCR MasterMix kit manufactured by Tiangen Biochemical technology Ltd. The PCR reaction system is as follows: to a PCR reaction system with a total volume of 50. mu.L, 25. mu.L of 2xTaq Plus PCR Master Mix, 2.5. mu.L (10. mu.M) of the forward primer PF, 2.5. mu.L (10. mu.M) of the reverse primer PR, 1. mu.L of the template were added, and sterilized distilled water was added to 50. mu.L. PCR reaction procedure: (1) pre-denaturation at 94 ℃ for 3min, (2) denaturation at 94 ℃ for 30sec, (3) annealing at 55 ℃ for 30sec, (4) extension at 72 ℃ for 3.5min, and steps (2) - (4) are performed for 30 cycles in total. The PCR product was stored at 4 ℃. After the PCR was completed, the sample was sampled and subjected to agarose gel electrophoresis to examine the amplification result, and the gel concentration was 1%. And cutting and recovering the gel, and treating the gel by using a gel recovery kit produced by Tiangen Biotechnology limited company (the process is carried out according to the instruction) to obtain a PCR amplification product, namely the trypsin target gene. Storing at-20 deg.C.

3) Obtaining of pET-28a (+) vector

Escherichia coli, which had been purchased from a company and stored with the plasmid pET28a, was activated in LB medium containing kanamycin, subcultured, and when the OD reached about 1.0, plasmid extraction was carried out using a plasmid miniprep kit (the extraction procedure was carried out as described above), to obtain a pET-28a (+) expression vector, and the next enzymatic reaction was carried out.

4) Plasmid construction

After the trypsin target gene and the pET-28a (+) expression vector are obtained, double distilled water, endonuclease buffer solution, enzyme digestion substrate and restriction enzyme (EcoR I and HindIII purchased from NEB company, specifications and methods are used and carried out according to the instructions) are respectively added into a PCR small tube for double enzyme digestion reaction, and the adding sequence is from a few to a few. The target gene and pET-28a (+) are subjected to double enzyme digestion at 37 ℃.

A trypsin gene fragment double enzyme digestion system: 11. mu.L of ultrapure water, 1. mu.L of HindIII, 1. mu.L of EcoR I, 3. mu.L of Buffer, and 14. mu.L of the trypsin target gene.

pET-28a (+) double enzyme system: pET-28a (+) plasmid 43. mu.L, Hind III 1. mu.L, EcoR I1. mu.L, Buffer 5. mu.L.

Performing double enzyme digestion reaction for 3h at 37 ℃ by using Hind III and EcoR I restriction enzymes, adding a Loading Buffer to terminate the reaction, and purifying and recovering the double enzyme digestion product according to the instruction of a glue recovery kit produced by Tiangen Biochemical technology limited company.

After the double-enzyme digestion products are cut by the same restriction enzyme, the double-enzyme digestion products have the same cohesive end and can be connected into a complete plasmid through DNA ligase. Putting the trypsin gene containing the same cohesive end and the pET-28a (+) double enzyme digestion product into the same PCR tubule, and adopting a 10 mu L system for the connection reaction: mu.L of the enzyme-cleaved product of the objective gene, 1. mu.L of the plasmid-cleaved product of pET-28a (+), 1. mu. L T4 DNA ligase (purchased from NEB) and 5. mu.L of ultrapure water were mixed well and ligated at 16 ℃ overnight. The successfully ligated plasmid was designated pET28a-Typ and the vector map is shown in FIG. 1.

5) Transformation of

(1) Coli E.coli BL21(DE3) competent cell preparation

A. Preparing LB slant, taking acceptor colibacillus to inoculate and activate, and culturing overnight at 37 ℃.

B. The activated recipient Escherichia coli colony is inoculated into LB culture medium, and is subjected to constant temperature shaking culture at 37 ℃ and 150rpm overnight.

C. The overnight cultured bacteria are taken by a pipette under the aseptic condition and inoculated into 50mL of fresh LB culture medium according to the inoculation amount of 1 percent, and the constant temperature shaking culture is carried out for 2-3 h at 37 ℃ and 150 rpm.

D. And (3) taking an uninoculated LB culture medium as a blank control, measuring the optical density value of the culture solution, and controlling the optical density value to be 0.2-0.5.

E. The culture was transferred into 50mL sterilized centrifuge tubes and placed on ice for 10 min.

F. Centrifuging at 4 deg.C and 4000rpm for 10min, recovering cells, and discarding supernatant.

G. 10mL of precooled 0.1M CaCl was added₂The solution was left on ice for 25 min.

H. Centrifuging at 4 deg.C and 4000rpm for 10min, recovering cells, and discarding supernatant.

I. 4mL of pre-cooled 0.1M CaCl containing 15% glycerol was added₂The solution suspends the cells, is placed on ice for 5min and is subpackaged with 200 mu L into Ep tubes, and is stored at-80 ℃ for standby.

(2) Coli BL21(DE3) competent cells, the transformation procedure was as follows:

a. the temperature of the thermostatic water bath was first adjusted to 42 ℃.

b. A tube (200. mu.l) of E.coli BL21(DE3) competent cells was taken out of an ultra-low temperature freezer at-80 ℃ and immediately warmed to melt with a finger, then inserted into ice and subjected to a 10min ice bath.

c. Mu.l of the ligation product pET28a-trypsin was added, gently shaken and then placed on ice for 20 min.

d. Shaking gently, inserting into 42 deg.C water bath, performing heat shock for 90s, rapidly placing back into ice, and standing for 5 min.

e. 900 μ L of antibiotic-free LB medium was added to the tube and gently mixed, and then fixed on a spring holder of a shaker, and shaken at 37 ℃ for 50 min.

f. To each of the solid LB plates containing kanamycin, 300. mu.l of the above-mentioned conversion mixture taken out from the clean bench was dropped, and the plate was uniformly coated with a glass coating rod which was burned with an alcohol lamp and cooled.

g. The marked culture dish is firstly placed in a constant temperature incubator at 37 ℃ for 30-60min, and after all the liquid on the surface permeates into the culture medium, the culture dish is placed in the constant temperature incubator at 37 ℃ for overnight in a reverse way.

And (3) carrying out recombinant plasmid identification on the grown single colony: and (3) selecting a single colony growing on the LB solid culture medium, inoculating the single colony into 5mL of LB liquid culture medium containing kanamycin, culturing overnight at 37 ℃ under 200rpm, taking part of bacterial liquid to preserve strains by using a glycerol tube, and taking a proper amount of bacterial liquid to extract recombinant plasmids according to the specification of a plasmid extraction kit. The extracted recombinant plasmid is subjected to double enzyme digestion by HindIII and EcoR I, the system is the same as that of pET-28a (+) double enzyme digestion, and the product after double enzyme digestion is subjected to electrophoresis analysis on a target gene fragment and a carrier fragment by using 1% agarose gel. And then, the recombinant plasmid identified as positive by double enzyme digestion is sent to Shanghai workers for sequence determination, and the next step of experiment is carried out if the determination is successful.

Example 2 expression and purification of Trypsin in E.coli

1) Protein expression

Inoculating the glycerol strain containing the recombinant plasmid into 6mL of LB liquid culture medium (containing 50mg/L kanamycin sulfate), and culturing at 37 ℃ and 220rpm overnight for 14 h;

transferring the overnight-cultured seeds to 500mL LB liquid medium at a ratio of 1:100, culturing at 37 ℃ and 150rpm to OD₆₀₀＝0.5；

Inducing the expression of the target gene on the recombinant plasmid by 0.5mM IPTG, and culturing overnight at the temperature of 18 ℃ and the rpm of 24 hours after induction;

centrifuging at 4000g and 4 ℃ for 5min to collect thalli, re-suspending the thalli by using 30mL of preservation buffer solution, and then crushing the thalli by using an ultrasonic crusher (working for 3s, interval of 7s, time of 10min and ultrasonic power of about 200W);

the crushed cell suspension is centrifuged at 12,000rpm and 4 ℃ for 30min, and the obtained supernatant is crude protein enzyme solution which can be used for enzyme activity test and subsequent protein purification experiments.

LB culture medium: 10g/L tryptone, 10g/L sodium chloride and 5g/L yeast extract powder.

Preservation buffer solution: 20mM Tris-HCl, 300mM NaCl, 10% glycerol, pH 8.0.

2) Protein purification

The target protein is fused with His label at C end, so the protein can be purified by affinity chromatography. The invention adopts the Ni-NTA purification medium of Shanghai work to purify the protein.

Step 1, adding 6mL of Ni-NTA purification medium into a 50mL empty chromatographic column, allowing the medium to freely settle, and draining the stock solution;

step 2, adding 4 times of column volume of washing buffer solution to balance the chromatography medium;

step 3, loading a clarified sample containing His-tagged protein into the column, and controlling the flow rate to be 0.5 mL/min;

step 4, washing the column by using a washing buffer solution with the flow rate of 1mL/min, wherein the flow rate is 10 times of the column volume so as to remove the foreign proteins;

step 5, eluting with 5 times of column volume elution buffer solution at the flow rate of 0.5-1mL/min, and collecting eluent;

and 6, collecting eluent, desalting and concentrating the purified protein by using a Millipore ultrafiltration tube (15mL of 10kDa), putting 10mL of eluent into the ultrafiltration tube, collecting the lower part of the ultrafiltration tube, centrifuging the lower part of the ultrafiltration tube at 4 ℃ and 3500rpm for 20min, reserving about 1.5mL of protein in the upper ultrafiltration tube, and reserving a buffer solution containing imidazole in the lower part of the ultrafiltration tube. And (3) pouring out the solution in the collection tube, then re-using the preservation buffer solution to fix the volume to 10mL, repeating the steps for 3 times, and sucking out the protein in the ultrafiltration tube by using a pipette gun.

Washing buffer solution: 10mM imidazole, 20mM Tris-HCl, 300mM NaCl, 10% glycerol, pH 8.0.

Elution buffer: 200mM imidazole, 20mM Tris-HCl, 300mM NaCl, 10% glycerol, pH 8.0.

EXAMPLE 3 Trypsin content determination

The Protein Content is detected by using BCA Protein Content Assay Kit (BCA Protein Content detection Kit, Beijing Box Biotechnology Co., Ltd.), and the detection method is as follows:

(1) sample dilution: diluting the sample by a proper time;

(2) preparing a BCA working solution: according to the usage amount, the BCA Solution: copper Solution 50:1(V: V) was formulated as BCA.

(3) Preparation of protein standard diluent: 100 μ L BSA Standard Solution was diluted to 1000 μ L with PBS Diluent, which was a 0.5mg/mL protein Standard dilution. Gradient dilutions were performed as in Table 1 using 0.5mg/mL protein standard dilutions.

TABLE 1

Numbering	1	2	3	4	5	6
							Standard dilution of protein (uL)	0	40	80	120	160	200
PBSDiluent(μL)	200	160	120	80	40	0
							Final protein concentration (mg/mL)	0	0.1	0.2	0.3	0.4	0.5

And (3) light absorption value determination: the absorbance values of the measurement tube and the standard tube at 562nm were measured and recorded as A measurement and A standard. Preparation and use of the standard curve: taking 0, 0.1, 0.2, 0.3, 0.4 and 0.5mg/mL as an abscissa (x) and the corresponding A standard as an ordinate (y) to obtain a linear regression equation, substituting the A measurement into a formula to calculate x (mg/mL), and multiplying the x (mg/mL) by the corresponding dilution factor to obtain the protein concentration of the sample. The protein concentration value of the enzyme solution was calculated as C (mg/mL) and used to calculate the trypsin specific activity.

Example 4 Trypsin Activity assay

The measurement was carried out by the ultraviolet absorption method using benzoyl-L-arginine ethyl ester (BAEE) as a substrate. The ultraviolet light absorption of benzoyl-L-arginine ethyl ester at the wavelength of 253nm is far weaker than that of benzoyl-L-arginine (BA). Under the catalysis of trypsin, benzoyl-L-arginine is gradually increased along with the hydrolysis of ester bonds, the ultraviolet light absorption of a reaction system is correspondingly increased, and the activity of the trypsin is calculated by delta A253 nm.

2 quartz cuvettes with covers and an optical path of 1cm were taken and 2.8mL of substrate solution preheated to 25 ℃ were added, respectively. 0.2mL of 0.01M HCl was added to a cuvette, and the solution in the cuvette was used as a blank solution to correct the zero absorbance of the spectrophotometer at 253 nm. And adding 0.2mL of enzyme solution to be detected into the other cuvette, immediately mixing uniformly, timing, reading once every half minute, and reading for 3-4 min. The delta A253 nm/min is controlled to be between 0.05 and 0.100.

And (3) drawing an enzymatic reaction kinetic curve, and solving the change rate of the absorbance at the reaction starting point along with time from the curve: delta A253_nmThe trypsin activity units are calculated as follows.

The enzyme activity unit is defined as: delta A_253nmThe increase of the enzyme activity per minute is 0.001, and the required enzyme amount is one enzyme activity unit U.

The enzyme activity (U/mL) of the trypsin solution is 5x ((. DELTA.A)_{253 nm}Min)/0.001) x dilution times trypsin specific activity (U/mL)/C of trypsin enzyme solution

Wherein C is the protein concentration (mg/mL) of the enzyme solution

The specific enzyme activity of the initial trypsin at 37 ℃ is 1862U/mg.

Example 5 mutation of enzyme construction of higher enzyme Activity of Trypsin

1) Error prone PCR

And (3) carrying out error-prone PCR by taking the synthesized primers PF and PR as primers and the synthesized sequence SEQ ID NO.2 as a template. An error-prone PCR kit (Beijing Tianenzze technologies, Inc.) was used.

The error-prone PCR reaction system is as follows: error-prone PCR Mix 3.0 μ L, error-prone PCR dNTP 3.0 μ L, MnCl₂3.0. mu.L (5mM), 1. mu.L of DNA template (10. mu.g/. mu.L), 1. mu.L of 10. mu.M PF, 1. mu.L of 10. mu.M PR, 1. mu.L of Taq DNA polymerase, and 17. mu.L of sterile double distilled water.

The error-prone PCR reaction procedure was as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 deg.C for 1min, annealing at 45 deg.C

1min, extension at 72 ℃ for 4min, and 32 cycles; extension at 72 ℃ for 10 min.

And after the amplification is finished, performing agarose gel electrophoresis and gel cutting recovery to obtain an error-prone PCR product.

2) Cleavage, ligation and transformation

And respectively adding double distilled water, an endonuclease buffer solution, an enzyme digestion substrate and a restriction endonuclease into the PCR tubule to perform double enzyme digestion reaction, wherein the adding sequence is from a few to a few. The error-prone PCR product and pET-28a (+) were subjected to double digestion at 37 ℃.

The error-prone PCR product double enzyme digestion reaction system is as follows: 11. mu.L of ultrapure water, 1. mu.L of HindIII, 1. mu.L of EcoR I, 3. mu.L of Buffer, and 14. mu.L of error-prone PCR product.

pET-28a (+) double enzyme system: pET-28a (+) plasmid 43. mu.L, Hind III 1. mu.L, EcoR I1. mu.L, Buffer 5. mu.L.

Performing double enzyme digestion reaction for 3h at 37 ℃ by using Hind III and EcoR I restriction enzymes, adding a Loading Buffer to terminate the reaction, and purifying and recovering the double enzyme digestion product according to the instruction of a glue recovery kit produced by Tiangen Biochemical technology limited company.

The target gene containing the same cohesive end and the pET-28a (+) double enzyme digestion product are placed in the same PCR tubule, and the ligation reaction adopts a 10 mu L system: mu.L of the enzyme-cleaved product of the objective gene, 1. mu.L of the plasmid-cleaved product of pET-28a (+), 1. mu. L T4 DNA ligase (purchased from NEB) and 5. mu.L of ultrapure water were mixed well and ligated at 16 ℃ overnight. The successfully ligated plasmid was designated pET28 a-EROR.

3) Expression, purification, content determination and enzyme activity determination of error-prone PCR product

The expression and purification of error-prone PCR products are the same as in example 2, the content measurement is the same as in example 3, and the enzyme activity measurement is the same as in example 4.

4) Screening and sequencing

Screening to obtain a strain clone with the specific enzyme activity of 2436U/mg at 37 ℃. Extracting plasmid, sequencing to obtain the coding sequence of trypsin shown in SEQ ID No.5 (with restriction enzyme cutting site and histidine tag).

gaattcATGACCCAATACCATCGTCGTGCACGTCTGCCACCTGTATCTCCTCGTGCAGATGATGGTGGTATGCGTCTGCTGGTGCTGTTCCTGCTGTTTATGGTTGAAGTCTGTGTTCAGTCTGCTGTAGCTCCGTCTCACCTGCAGCATCTGCTGTCTTCCCGTATCGTTGGTGGTGAAGACGCTGTTCAGGGCGAGTTCCCTTCTCAGGTGAGCATCGAACATGTTTCTTCTCTGGGCCGTAACCATATCTGCGGCGGCATTGTGCTGAGCGCGACCGATGTACTGACTGCAGCGCACTGTATCTCTCGCTTCAGCGAACACAACATCGACATTGTTGCCGGTATCAACCGTCTGAGCGACACCTCTTCCAACCGTCAGACCGTGGAAGCAGTTCACTTCATCATCCACGAAGATTTCAACGACATTACTATGGGTAGCGACATTGCAATTATCCGCCTGAAACACGCGCTGGAACTGAACGAATGGGTCCAGCCGGTCGTTATGCCGAAAACCGGCGAAACCGCACAAGAAGGTGAAATGTGTACTGTAGTTGGCTGGGGTGCGACCTTCGAGGATGGTGATCTGTCCGACGTTCTGCAGAAAGTTTCCCTGCCAATTCAGTCTGATAGCTACTGCCGTTCTGCCTACGGTTACTCTGCGGTCGAAGATTCTATGCTGTGTGCAGGCTCTCCTGAAGGTGGTGCTGATGCCTGTGATGGTGATCGTGGTGGCCCGATGCTGTGTCGTGGTCACCTGCACGGTCTGTCTTCTTGGGGTGAAGGCTGCGGTCATAGCTTTTATCCGGGTGTCTATACTGAGGTAAGCGCTTTCCTGCACTGGAACCCGAAAAGCACCGAAGACCTGGAGCTGGAAGCTCTGCTGGGTGATGACTCTGCTGCACCGGAGGAAACCCCGTCTGATGACCCGGAAGTACAGGTGGTTGTTGCGGAATCTCCGGAAGACCATGATGTTCCGCGCGAACTGCCGCCGGAAGTGCACCACCACCACCACCACTAAaagctt；SEQ ID NO.5。

The corresponding amino acid sequence is shown in SEQ ID NO. 6.

mtqyhrrarlppvspraddggmrllvlfllfmvevcvqsavapshlqhllssrivggedavqgefpsqvsiehvsslgrnhicggivlsatdvltaahcisrfsehnidivaginrlsdtssnrqtveavhfiihedfnditmgsdiaiirlkhalelnewvqpvvmpktgetaqegemctvvgwgatfedgdlsdvlqkvslpiqsdsycrsaygysavedsmlcagspeggadacdgdrggpmlcrghlhglsswgegcghsfypgvytevsaflhwnpkstedleleallgddsaapeetpsddpevqvvvaespedhdvprelppevhhhhhh；SEQ ID NO.6。

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Zhongnong Huawei biopharmaceutical (Hubei) Co., Ltd

<120> construction method of high-activity traditional Chinese medicine feed additive trypsin

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 331

<212> PRT

<213> Artificial Sequence

<400> 1

Met Thr Gln Tyr His Arg Arg Ala Arg Leu Pro Pro Val Ser Pro Arg

1 5 10 15

Ala Asp Asp Gly Gly Met Arg Leu Leu Val Leu Phe Leu Leu Phe Met

20 25 30

Val Glu Val Cys Val Gln Ser Ala Val Ala Pro Ser His Leu Gln His

35 40 45

Leu Leu Ser Ser Arg Ile Val Gly Gly Glu Asp Ala Val Gln Gly Glu

50 55 60

Phe Pro Phe Gln Val Ser Ile Glu His Val Gly Ile Leu Gly Arg Asn

65 70 75 80

His Ile Cys Gly Gly Ile Val Leu Ser Ala Thr Asp Val Leu Thr Ala

85 90 95

Ala His Cys Ile Ser Arg Phe Ser Glu His Asn Ile Asp Ile Val Ala

100 105 110

Gly Ile Asn Arg Leu Ser Asp Thr Ser Ser Asn Arg Gln Thr Val Glu

115 120 125

Ala Val His Phe Ile Ile His Glu Asp Phe Asn Asp Ile Thr Met Gly

130 135 140

Ser Asp Ile Ala Ile Ile Arg Leu Lys His Ala Leu Glu Leu Asn Glu

145 150 155 160

Trp Val Gln Pro Val Val Met Pro Lys Thr Gly Glu Thr Ala Gln Glu

165 170 175

Gly Glu Met Cys Thr Val Val Gly Trp Gly Ala Thr Phe Glu Asp Gly

180 185 190

Asp Leu Ser Asp Val Leu Gln Lys Val Ser Val Pro Ile Gln Ser Asp

195 200 205

Ser Tyr Cys Arg Ser Ala Tyr Gly Tyr Ser Ala Val Glu Asp Ser Met

210 215 220

Leu Cys Ala Gly Ser Pro Glu Gly Gly Ala Asp Ala Cys Asp Gly Asp

225 230 235 240

Arg Gly Gly Pro Met Leu Cys Arg Gly His Leu His Gly Ile Ser Ser

245 250 255

Trp Gly Glu Gly Cys Gly His Ser Phe Tyr Pro Gly Val Tyr Thr Glu

260 265 270

Val Ser Ala Phe Leu His Trp Asn Pro Lys Ser Thr Glu Asp Leu Glu

275 280 285

Leu Glu Ala Leu Leu Gly Asp Asp Ser Ala Ala Pro Glu Glu Thr Pro

290 295 300

Ser Asp Asp Pro Glu Val Gln Val Val Val Ala Glu Ser Pro Glu Asp

305 310 315 320

His Asp Val Pro Arg Glu Leu Pro Pro Glu Val

325 330

<210> 2

<211> 1026

<212> DNA

<213> Artificial Sequence

<400> 2

gaattcatga cccaatacca tcgtcgtgca cgtctgccac ctgtatctcc tcgtgcagat 60

gatggtggta tgcgtctgct ggtgctgttc ctgctgttta tggttgaagt ctgtgttcag 120

tctgctgtag ctccgtctca cctgcagcat ctgctgtctt cccgtatcgt tggtggtgaa 180

gacgctgttc agggcgagtt ccctttccag gtgagcatcg aacatgttgg tattctgggc 240

cgtaaccata tctgcggcgg cattgtgctg agcgcgaccg atgtactgac tgcagcgcac 300

tgtatctctc gcttcagcga acacaacatc gacattgttg ccggtatcaa ccgtctgagc 360

gacacctctt ccaaccgtca gaccgtggaa gcagttcact tcatcatcca cgaagatttc 420

aacgacatta ctatgggtag cgacattgca attatccgcc tgaaacacgc gctggaactg 480

aacgaatggg tccagccggt cgttatgccg aaaaccggcg aaaccgcaca agaaggtgaa 540

atgtgtactg tagttggctg gggtgcgacc ttcgaggatg gtgatctgtc cgacgttctg 600

cagaaagttt ccgttccaat tcagtctgat agctactgcc gttctgccta cggttactct 660

gcggtcgaag attctatgct gtgtgcaggc tctcctgaag gtggtgctga tgcctgtgat 720

ggtgatcgtg gtggcccgat gctgtgtcgt ggtcacctgc acggtatctc ttcttggggt 780

gaaggctgcg gtcatagctt ttatccgggt gtctatactg aggtaagcgc tttcctgcac 840

tggaacccga aaagcaccga agacctggag ctggaagctc tgctgggtga tgactctgct 900

gcaccggagg aaaccccgtc tgatgacccg gaagtacagg tggttgttgc ggaatctccg 960

gaagaccatg atgttccgcg cgaactgccg ccggaagtgc accaccacca ccaccactaa 1020

aagctt 1026

<210> 3

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 3

cggaattcat gacccaatac catcgtcgt 29

<210> 4

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 4

ccaagctttt agtggtggtg gtggtggt 28

<210> 5

<211> 1026

<212> DNA

<213> Artificial Sequence

<400> 5

gaattcatga cccaatacca tcgtcgtgca cgtctgccac ctgtatctcc tcgtgcagat 60

gatggtggta tgcgtctgct ggtgctgttc ctgctgttta tggttgaagt ctgtgttcag 120

tctgctgtag ctccgtctca cctgcagcat ctgctgtctt cccgtatcgt tggtggtgaa 180

gacgctgttc agggcgagtt cccttctcag gtgagcatcg aacatgtttc ttctctgggc 240

cgtaaccata tctgcggcgg cattgtgctg agcgcgaccg atgtactgac tgcagcgcac 300

tgtatctctc gcttcagcga acacaacatc gacattgttg ccggtatcaa ccgtctgagc 360

gacacctctt ccaaccgtca gaccgtggaa gcagttcact tcatcatcca cgaagatttc 420

aacgacatta ctatgggtag cgacattgca attatccgcc tgaaacacgc gctggaactg 480

aacgaatggg tccagccggt cgttatgccg aaaaccggcg aaaccgcaca agaaggtgaa 540

atgtgtactg tagttggctg gggtgcgacc ttcgaggatg gtgatctgtc cgacgttctg 600

cagaaagttt ccctgccaat tcagtctgat agctactgcc gttctgccta cggttactct 660

gcggtcgaag attctatgct gtgtgcaggc tctcctgaag gtggtgctga tgcctgtgat 720

ggtgatcgtg gtggcccgat gctgtgtcgt ggtcacctgc acggtctgtc ttcttggggt 780

gaaggctgcg gtcatagctt ttatccgggt gtctatactg aggtaagcgc tttcctgcac 840

tggaacccga aaagcaccga agacctggag ctggaagctc tgctgggtga tgactctgct 900

gcaccggagg aaaccccgtc tgatgacccg gaagtacagg tggttgttgc ggaatctccg 960

gaagaccatg atgttccgcg cgaactgccg ccggaagtgc accaccacca ccaccactaa 1020

aagctt 1026

<210> 6

<211> 337

<212> PRT

<213> Artificial Sequence

<400> 6

Met Thr Gln Tyr His Arg Arg Ala Arg Leu Pro Pro Val Ser Pro Arg

1 5 10 15

Ala Asp Asp Gly Gly Met Arg Leu Leu Val Leu Phe Leu Leu Phe Met

20 25 30

Val Glu Val Cys Val Gln Ser Ala Val Ala Pro Ser His Leu Gln His

35 40 45

Leu Leu Ser Ser Arg Ile Val Gly Gly Glu Asp Ala Val Gln Gly Glu

50 55 60

Phe Pro Ser Gln Val Ser Ile Glu His Val Ser Ser Leu Gly Arg Asn

65 70 75 80

His Ile Cys Gly Gly Ile Val Leu Ser Ala Thr Asp Val Leu Thr Ala

85 90 95

Ala His Cys Ile Ser Arg Phe Ser Glu His Asn Ile Asp Ile Val Ala

100 105 110

Gly Ile Asn Arg Leu Ser Asp Thr Ser Ser Asn Arg Gln Thr Val Glu

115 120 125

Ala Val His Phe Ile Ile His Glu Asp Phe Asn Asp Ile Thr Met Gly

130 135 140

Ser Asp Ile Ala Ile Ile Arg Leu Lys His Ala Leu Glu Leu Asn Glu

145 150 155 160

Trp Val Gln Pro Val Val Met Pro Lys Thr Gly Glu Thr Ala Gln Glu

165 170 175

Gly Glu Met Cys Thr Val Val Gly Trp Gly Ala Thr Phe Glu Asp Gly

180 185 190

Asp Leu Ser Asp Val Leu Gln Lys Val Ser Leu Pro Ile Gln Ser Asp

195 200 205

Ser Tyr Cys Arg Ser Ala Tyr Gly Tyr Ser Ala Val Glu Asp Ser Met

210 215 220

Leu Cys Ala Gly Ser Pro Glu Gly Gly Ala Asp Ala Cys Asp Gly Asp

225 230 235 240

Arg Gly Gly Pro Met Leu Cys Arg Gly His Leu His Gly Leu Ser Ser

245 250 255

Trp Gly Glu Gly Cys Gly His Ser Phe Tyr Pro Gly Val Tyr Thr Glu

260 265 270

Val Ser Ala Phe Leu His Trp Asn Pro Lys Ser Thr Glu Asp Leu Glu

275 280 285

Leu Glu Ala Leu Leu Gly Asp Asp Ser Ala Ala Pro Glu Glu Thr Pro

290 295 300

Ser Asp Asp Pro Glu Val Gln Val Val Val Ala Glu Ser Pro Glu Asp

305 310 315 320

His Asp Val Pro Arg Glu Leu Pro Pro Glu Val His His His His His

325 330 335

His

Claims (2)

1. A construction method of a high-activity traditional Chinese medicine feed additive trypsin is characterized by comprising the following specific steps:

(1) carrying out error-prone PCR by using SEQ ID NO.2 as a template and PF and PR as primers;

the primer sequences are as follows:

PF：5’-cggaattcATGACCCAATACCATCGTCGT-3’；SEQ ID NO.3；EcoR I；

PR：5’-ccaagcttTTAGTGGTGGTGGTGGTGGT-3’；SEQ ID NO.4；Hind III；

the error-prone PCR reaction system is as follows: error-prone PCR Mix 3.0 μ L, error-prone PCR dNTP 3.0 μ L, 5mM MnCl₂3.0 μ L, 10 μ g/μ L DNA template 1 μ L, 10 μ M PF 1 μ L, 10 μ M PR1 μ L, Taq DNA polymerase 1 μ L, sterilized double distilled water 17 μ L;

the error-prone PCR reaction procedure was as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 deg.C for 1min, annealing at 45 deg.C for 1min, extension at 72 deg.C for 4min, and 32 cycles; extending for 10min at 72 ℃;

(2) carrying out agarose gel electrophoresis and gel recovery on the error-prone PCR product;

(3) and carrying out double enzyme digestion, connection and transformation on the recovered error-prone PCR product and pET-28a (+) to E.coli BL21(DE3) competent cells, and carrying out expression and purification to obtain the high-activity trypsin, wherein the amino acid sequence of the trypsin is shown in SEQ ID No. 6.

2. The method for constructing trypsin as a highly active Chinese medicinal feed additive according to claim 1, wherein in step (3), the protein is purified by using Ni-NTA purification medium.

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