CN105237627A - Purification method of low-molecular-weight bovine lactoferricin antimicrobial peptide - Google Patents

Abstract

The invention relates to a purification method of low-molecular-weight bovine lactoferricin antimicrobial peptide. According to the method, amino acid coded sequence No. 1 and nucleotide coded sequence No. 2 of bovine lactoferricin LfcinB are coded, a carrier pPIC9K-LfcinB is built and expressed, the carrier is converted into a pichia pastoris GS115 host cell, and an expressible reconstitution cell is formed; the host cell is cultured and expresses LfcinB antimicrobial peptide; mother liquid concentration is separated, protein inhibitor and a protective agent are added, electrophoresis is conducted with 20% tri(hydroxymethyl)methylglycine-lauryl sodium sulfate-polyacrylamide gel through dialysis, objective protein with higher purity is obtained, and higher antibacterial activity is achieved through detection.

Description

A kind of purification process of lower molecular weight Bovine lactoferricin antibacterial peptide

Technical field

The present invention relates to a kind of purification process of lower molecular weight Bovine lactoferricin antibacterial peptide, belong to technological field of biochemistry.

Background technology

Antibiotic discovery and application has saved countless life, also brings huge Social benefit and economic benefit simultaneously.But in recent years, in clinical treatment, the microbiotic of novel structure finds that plaque is weary, and the fast development of anti-medicine flora, makes some infectious diseases become a difficult problem for clinical treatment.Antibacterial skin self-discovery and research so far, have been considered to existing antibiotic best substitute, its Study and appliance oneself become study hotspot in field of biological pharmacy.Why the research of antibacterial skin gains great popularity is because the following features itself possessed: 1. relative molecular mass is less; 2. stable in physicochemical property; 3. has a broad antifungal spectrum, especially antagonistic drug bacterium has significant curative effect; 4. self not easily produces resistance again.

Because the natural resource of antibacterial skin are limited, thus in microorganism, directly the Main Means obtaining antibacterial skin Biao Da do not become by antibacterial peptide gene by genetically engineered.Engineered product composition is complicated, higher to the purity requirement of product, and in the middle of the process of purifying, there is the trend, particularly some low-molecular-weight albumen of assembling sex change, larger than general protein purification difficulty.And by adding some protective materials; can make albumen avoid degraded, by dialysis, 20% trishydroxymethyl glycine-sodium lauryl sulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) running gel electrophoresis can be separated fast and effectively and obtain low-molecular-weight albumen.

Summary of the invention

The object of the invention is to, a kind of purification process of lower molecular weight Bovine lactoferricin antibacterial peptide is provided, the method is according to the amino acid coding NO.1 of coding Bovine lactoferricin LfcinB and corresponding nucleotide coding sequence NO.2, construction of expression vector pPIC9K-LfcinB, and by vector to Pichia pastoris GS115 host cell, form effable reconstitution cell; Cultivate host cell, make it express LfcinB antibacterial peptide; Be separated concentrated mother liquor; add protein inhibitor and protective material; through dialysis and 20% trishydroxymethyl glycine-sodium lauryl sulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) electrophoresis; obtain the target protein that purity is higher, there is higher bacteriostatic activity after testing.

The purification process of a kind of lower molecular weight Bovine lactoferricin antibacterial peptide of the present invention, the method relates to the amino acid coding NO.1:Ac-FKCRRWQWRMKKLGAP-NH2 of Bovine lactoferricin LfcinB; Nucleotide sequence NO.2 :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', the upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 ' of Bovine lactoferricin LfcinB coding; Concrete operations follow these steps to carry out:

The gene expression system of the Bovine lactoferricin described in structure:

A, according to Bovine lactoferricin LfcinB aminoacid sequence No1:Ac-FKCRRWQWRMKKLGAP-NH2; Design corresponding nucleotide sequence No2 :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 ', by the restriction enzyme site of the upstream in Bovine lactoferricin LfcinB gene reading code district with SnaB I, downstream is with NotI restriction enzyme site;

B, first fragment to be loaded in pMD19T, then cut through enzyme, object fragment is loaded pPIC9K, obtains the expression vector built;

C, the expression vector built is transformed in pichia spp, screens positive colony, after PCR qualification is correct, cultivate;

D, the positive colony screened are cultivated at 50 milliliters of BMGY, temperature 30 DEG C, 200rpm shaking culture absorbancy OD ₂₀₀for 2-6OD value, the centrifugal 5min of 3000rpm, discards substratum, again cultivates with 1 liter of BMGY substratum, and adds methanol induction expression target protein;

The purifying of Bovine lactoferricin antibacterial peptide:

E, cultivate 72h, rapidly nutrient solution is placed on ice, after frozen centrifugation, collect supernatant liquor, with the resuspended thalline of the phosphoric acid buffer of pH6.8, add lyase 100mg/100ml, room temperature keeps 30min, 3000rmp is centrifugal, collect supernatant liquor, twice supernatant liquor is merged, add phenylmethylsulfonyl fluoride 10mM, supernatant concentration 10 times will be expressed with trichloroacetic acid precipitation, again dissolve with the phosphoric acid buffer of pH6.8, with the trishydroxymethyl glycine-sodium lauryl sulphate-polyacrylamide gel electrophoresis of 20%, target protein detected, the filter membrane of the albumen 10Kd after dissolving is dialysed, target protein enters in solution, then molecular weight cut-off filtrate is selected to be that the dialysis membrane of 1000Da retains desired polypeptides except freshen.

Adopt plate doubling dilution to measure anti-microbial activity the lower molecular weight Bovine lactoferricin antibacterial peptide obtained after purifying and detect biological activity.

The purification process of a kind of lower molecular weight Bovine lactoferricin antibacterial peptide of the present invention, provides Bovine lactoferricin (LfcinB) aminoacid sequence No1:Ac-FKCRRWQWRMKKLGAP-NH2 in the method; And the nucleotide sequence No2 of encoding gene :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 ', by the upstream in gene wherein LfcinB gene reading code district with the restriction enzyme site of SnaB I, downstream is with NotI restriction enzyme site.First fragment is loaded in pMD19T, then cut through enzyme, object fragment is loaded pPIC9K; The expression vector built is transformed into GS115, G418 screening positive clone in pichia spp;

The positive colony screened is cultivated at 50 milliliters of BMGY, 30 DEG C, and 200rpm shaking culture OD200 is the centrifugal 5min of 2-6OD, 3000rpm, discards substratum, again cultivates with 1LBMGY substratum, and adds methanol induction expression target protein;

Cultivate 72h, rapidly nutrient solution is placed on ice, after frozen centrifugation, collect supernatant liquor, with the resuspended thalline of the phosphoric acid buffer of PH6.8, add lyase 100mg/100ml, room temperature keeps 30min, 3000rmp is centrifugal, collect supernatant liquor, twice supernatant liquor is merged, adds phenylmethylsulfonyl fluoride (PMSF) 10mM, trichoroacetic acid(TCA) (TCA) precipitator method will express supernatant concentration 10 times, again dissolve with the phosphoric acid buffer of the PH6.8 containing 2% beta-mercaptoethanol (v/v); With trishydroxymethyl glycine-sodium lauryl sulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) electrophoresis of 20%, target protein detected; The filter membrane of the albumen 10Kd after dissolving is dialysed, target protein enters in solution, then filtrate selected molecular weight cut-off to be that the dialysis membrane of 1000Da retains desired polypeptides except freshen, polypeptide detects bacteriostatic activity, has obvious restraining effect to intestinal bacteria and gold-coloured staphylococci.

Accompanying drawing explanation

Fig. 1 is pPIC9K-LfcinB expression vector establishment figure of the present invention;

Fig. 2 is that pPIC9K-LfcinB positive colony enzyme of the present invention cuts detection figure;

Fig. 3 is the PCR detection figure of pPIC9K-LfcinB positive colony of the present invention;

Fig. 4 is that the trishydroxymethyl glycine-sodium lauryl sulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) of LfcinB of the present invention detects figure.

Embodiment

Embodiment

The gene expression system of the Bovine lactoferricin described in structure:

A, according to Bovine lactoferricin LfcinB aminoacid sequence No1:Ac-FKCRRWQWRMKKLGAP-NH2; Design corresponding nucleotide sequence No2 :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 ', by the restriction enzyme site of the upstream in Bovine lactoferricin LfcinB gene reading code district with SnaB I, downstream is with NotI restriction enzyme site;

B, first fragment to be loaded in pMD19T, then cut through enzyme, object fragment is loaded pPIC9K, obtains the expression vector built;

C, the expression vector built is transformed in pichia spp, screens positive colony, after PCR qualification is correct, cultivate;

D, the positive colony screened are cultivated at 50 milliliters of BMGY, temperature 30 DEG C, 200rpm shaking culture absorbancy OD ₂₀₀for 2-6OD value, the centrifugal 5min of 3000rpm, discards substratum, again cultivates with 1 liter of BMGY substratum, and adds methanol induction expression target protein;

The purifying of Bovine lactoferricin antibacterial peptide:

E, cultivate 72h, rapidly nutrient solution is placed on ice, after frozen centrifugation, collect supernatant liquor, with the resuspended thalline of the phosphoric acid buffer of pH6.8, add lyase 100mg/100ml, room temperature keeps 30min, 3000rmp is centrifugal, collect supernatant liquor, twice supernatant liquor is merged, add phenylmethylsulfonyl fluoride (PMSF) 10mM, supernatant concentration 10 times will be expressed with trichloroacetic acid precipitation, again dissolve with the phosphoric acid buffer of pH6.8, with trishydroxymethyl glycine-sodium lauryl sulphate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) gel electrophoresis of 20%, target protein detected, the filter membrane of the albumen 10Kd after dissolving is dialysed, target protein enters in solution, then molecular weight cut-off filtrate is selected to be that the dialysis membrane of 1000Da retains desired polypeptides except freshen.

Adopt plate doubling dilution to measure anti-microbial activity the lower molecular weight Bovine lactoferricin antibacterial peptide obtained after purifying and detect biological activity.In table 1:

LfcinB bacteriostatic activity after table 1 purifying detects

As can be seen from the table, when product is not purified, anti-microbial activity is low, and after purifying, anti-microbial activity improves.It can also be seen that there is good inhibit activities to the bacterial strain (1515,12-8) developed immunity to drugs clinically at present from upper table.

Title: a kind of purification process of lower molecular weight Bovine lactoferricin antibacterial peptide

Applicant: Chinese Academy Of Sciences Xinjiang physics & chemistry Technology Research Institute

The gene order LfcinB of Bovine lactoferricin, amino acid coding NO.1:Ac-FKCRRWQWRMKKLGAP-NH2.

The gene order LfcinB of Bovine lactoferricin, nucleotide coding sequence NO.2 :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 '.

Claims (1)

1. a purification process for lower molecular weight Bovine lactoferricin antibacterial peptide, is characterized in that the method relates to the amino acid coding NO.1:Ac-FKCRRWQWRMKKLGAP-NH2 of Bovine lactoferricin LfcinB; Nucleotide sequence NO.2 :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', the upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 ' of Bovine lactoferricin LfcinB coding; Concrete operations follow these steps to carry out:

The gene expression system of the Bovine lactoferricin described in structure:

A, according to Bovine lactoferricin LfcinB aminoacid sequence No1:Ac-FKCRRWQWRMKKLGAP-NH2; Design corresponding nucleotide sequence No2 :-TCAAATGCTGGCGTTGGCAGTGGCGTTGGAAGAAATTGGGTGCTCCTTTTC-3 ' downstream 5 ', upstream 5 '-GAAAGCACGACGCACGCAAGTGATAGAAGGAGCACCCAATTTCTTCCA-3 ', by the restriction enzyme site of the upstream in Bovine lactoferricin LfcinB gene reading code district with SnaB I, downstream is with NotI restriction enzyme site;

B, first fragment to be loaded in pMD19T, then cut through enzyme, object fragment is loaded pPIC9K, obtains the expression vector built;

C, the expression vector built is transformed in pichia spp, screens positive colony, after PCR qualification is correct, cultivate;

D, the positive colony screened are cultivated at 50 milliliters of BMGY, temperature 30 DEG C, 200rpm shaking culture absorbancy OD ₂₀₀for 2-6OD value, the centrifugal 5min of 3000rpm, discards substratum, again cultivates with 1 liter of BMGY substratum, and adds methanol induction expression target protein;

The purifying of Bovine lactoferricin antibacterial peptide:

E, cultivate 72h, rapidly nutrient solution is placed on ice, after frozen centrifugation, collect supernatant liquor, with the resuspended thalline of the phosphoric acid buffer of pH6.8, add lyase 100mg/100ml, room temperature keeps 30min, 3000rmp is centrifugal, collect supernatant liquor, twice supernatant liquor is merged, add phenylmethylsulfonyl fluoride 10mM, supernatant concentration 10 times will be expressed with trichloroacetic acid precipitation, again dissolve with the phosphoric acid buffer of pH6.8, with the trishydroxymethyl glycine-sodium lauryl sulphate-polyacrylamide gel electrophoresis of 20%, target protein detected, the filter membrane of the albumen 10Kd after dissolving is dialysed, target protein enters in solution, then molecular weight cut-off filtrate is selected to be that the dialysis membrane of 1000Da retains desired polypeptides except freshen.