CN107236030A - A kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process - Google Patents

Abstract

The invention discloses a kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process, belong to Insect immunity field.Comprise the following steps：MRNA is extracted from silkworm fat-body, reverse transcription is into cDNA, cultivated silkworm peptidoglycan is expanded from cDNA by designing primer and recognizes the gene of Protein S 1, cultivated silkworm peptidoglycan is recognized into the gene cloning of Protein S 1 into PET 30a using the method for molecular cloning and BL21 (DE3) is converted.By BL21 (DE3) incubated overnight containing target gene, by 1:It is 0.5 or so that 100 dilution bacterium solutions, which shake bacterium to OD values, adds final concentration 0.8mMIPTG and 20% glucose induction 5h, and supernatant is abandoned in centrifugation, collects thalline.Prokaryotic expression system genetic map used in the present invention is clear and definite, it is easy to operate, and saves the time, being capable of high-caliber expression foreign protein.

Description

A kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process

Technical field

The invention belongs to Insect immunity field, more particularly to a kind of cultivated silkworm peptidoglycan identification albumen（PGRP-S1）Protokaryon Expression.

Background technology

Silkworm is a kind of important economic insects, and silk is used for making silk, sells at home and abroad, driven relevant enterprise Development, greatly facilitated the economic development of China.Contain abundant protein, edible in silkworm chrysalis.What silkworm excrement and silkworm sloughed off Micromicro is used as medicine, with important medical value.So in order to improve silkworm survival rate, enhancing silkworm is resisted to pathogenic microorganism Ability, immune research silkworm is essential.And had increasingly on the document that microbial pathogens is immunized silkworm It is reported more.

Silkworm belongs to lepidopterous insects, is both important economic insects, is important model organism again.Silkworm lacks the day after tomorrow It is immune, a set of fairly perfect immunologic mechanism i.e. congenital immunity is formd during the long-term evolution with microorganism, for The invasion of imperial pathogenic microorganism.Silkworm congenital immunity rely primarily on pattern recognition receptors identification pathogen associated molecular pattern and then Congenital immunity path is activated, so as to kill microorganism.These pathogen associated molecular patterns only exist pathogenic microorganism without depositing In host, such as lipopolysaccharides, peptide glycan, these pathogen associated molecular patterns are recognized by corresponding pattern recognition receptors, from And activate the signal path in downstream.Peptidoglycan recognition protein（PGRP）Family is the most important pattern recognition receptors of silkworm, and it can To recognize the peptide glycan of pathogenic microorganism, and then the immune pathway of silkworm is triggered, so as to activate or suppress the expression of antibacterial peptide.Have Research shows that silkworm infection Gram-negative bacteria activation Imd paths obtain immune response, gram-positive bacteria activation Imd paths Immune response is obtained, but also report shows to connect each other between both approach on evidence.

12 peptidoglycan identification protein genes are identified in the genome of silkworm, these peptidoglycan recognition proteins have one Individual PGRP domains, for combining the peptide glycan of pathogenic microorganism, and then start or suppress the synthesis of antibacterial peptide.Table on evidence Some bright peptidoglycan recognition proteins can also trigger the activation of phenol oxidase path, promote melanism effect.Some peptide glycans are recognized Albumen can also kill bacterium directly as bactericide.

The content of the invention

The invention provides a kind of structure of the prokaryotic expression carrier of cultivated silkworm peptidoglycan identification Protein S 1.

The present invention is realized by following technical proposal：

The method that cultivated silkworm peptidoglycan recognizes the prokaryotic expression of Protein S 1, extracts total serum IgE from silkworm fat-body tissue, passes through reverse transcription CDNA is synthesized, as template, expand from silkworm fat-body tissue by designing primer and obtains cultivated silkworm peptidoglycan identification albumen S1 genes.

The method that cultivated silkworm peptidoglycan recognizes the prokaryotic expression of Protein S 1,

The sense primer for designing primer is 5'- CGCAGGATCCGACATGGCCCGCCTCC -3'（BamH1）, （SEQ ID NO.1）；

Anti-sense primer is 5'- ATTAGCGGCCGCTCTTGATGGAGTCCACGTTCT -3'(Not1)（SEQ ID NO.2）. Restriction enzyme site is inserted in the primer of design, in order to which cultivated silkworm peptidoglycan is recognized into the gene cloning of Protein S 1 with the method for molecular cloning Into prokaryotic expression carrier PET-30a, the prokaryotic expression carrier built is converted into DH5 α, by bacterium solution PCR and digestion verification Positive colony sequencing, will be sequenced correct positive colony and extracts plasmid conversion BL21（DE3）.

The method that cultivated silkworm peptidoglycan recognizes the prokaryotic expression of Protein S 1, will be sequenced correct BL21（DE3）Incubated overnight, then With 1:100 are diluted with fresh culture, continue to cultivate to OD values about 0.5 or so, add final concentration of 0.8mM IPTG with Mass concentration is 20% glucose induction, and induction time is 5h.Because the albumen of expression carries His labels, nickel ion can be used Chelating magnetic bead is purified.

The method that nickel ion chelating magnetic bead carries out purifying inclusion body protein, is carried out as steps described below：

（1）The bacterium solution of induction is centrifuged, precipitation is collected, precipitation is washed twice with 1 × PBS, supernatant is abandoned in centrifugation, precipitation adds suitable 1 × PBS of amount simultaneously suspends, ultrasonic on ice（Power：25%, operation 3 seconds, interval 8 seconds, 25 times）Cracking, centrifugation, takes supernatant respectively 12%SDS-PAGE is carried out with precipitation, to determine the solubility of destination protein.

（2）Sample treatment：Precipitate with appropriate inclusion body cleaning solution（1M guanidine hydrochlorides, 20mM sodium phosphates, 500mMNaCl, PH =7.8）It is resuspended, supernatant is abandoned in centrifugation.Precipitate with appropriate inclusion body lysate（6M guanidine hydrochlorides, 20mM sodium phosphates, 500mMNaCl, PH=7.8）It is resuspended, centrifugation obtains supernatant.

（3）Magnetic bead is pre-processed：Use Buffer A（8M urea, 20mM sodium phosphates, 500mMNaCl, PH=7.8）Balance nickel from Son chelating magnetic bead magnetic bead is combined with destination protein：Added in magnetic bead in sample handling procedure and centrifuge obtained supernatant, make purpose Albumen is combined with magnetic bead.

（4）Magnetic bead is washed：Use Buffer B（8M urea, 20mM sodium phosphates, 500mMNaCl, PH=6.0）With Buffer C （8M urea, 20mM sodium phosphates, 500mMNaCl, PH=5.3）Magnetic bead is washed, with except foreigh protein removing.

（5）Destination protein is eluted：Use BufferD（8M urea, 20mM sodium phosphates, 500mMNaCl, PH=4.0）Elute purpose Albumen.

（6）Destination protein renaturation：Renaturation is carried out to inclusion body using dialysis.

In place of the advantage of the present invention：The tedious steps that albumen is extracted from Tissues of Silkworm Bombyx Moril are eliminated, experiment is greatly saved Cost and time.

Brief description of the drawings

Fig. 1 is that PCR is expanded in the result that cultivated silkworm peptidoglycan recognizes the gene of Protein S 1, figure：1-6:Using fat-body as template, ladder Spend PCR amplification S1 genes；

Fig. 2 is bacterium solution PCR results, in figure：1：DL5000DNA Marker；2-3：The bacterium solution PCR primer of one bacterium colony；3-4：Separately The bacterium solution PCR primer of one bacterium colony；

Fig. 3 is the double digestion result of recombinant plasmid, in figure：1：DL5000DNA Marker；2-3：The double digestion production of recombinant plasmid Thing；

Fig. 4 is induced for IPTG in the result of destination protein expression, figure：1：Albumen Marker；2：Bacterium solution is not induced；3：IPTG is induced Bacterium solution；

Fig. 5 is determines that destination protein whether there is in precipitation, in figure：1：Bacterium solution is not induced；2：The bacterium solution of IPTG inductions；3：It is super Total bacterium solution is taken after sonication；4：Supernatant is taken after ultrasonically treated；5：Precipitation is taken after ultrasonically treated；

Fig. 6 for purifying destination protein SDS-PAGE results, in figure：1：Albumen Marker；2：Bacterium solution is not induced；3：Before purification Albumen；4：The albumen of purifying；

Fig. 7 is the destination protein Western blot result figures of purifying, in：1：Bacterium solution is not induced；2：Albumen before purification；3： The albumen of purifying.

Embodiment

In order to be best understood from the purpose of the present invention and advantage, the present invention will be further explained in conjunction with the embodiments.Institute Only it is used for explaining the present invention for embodiment, but does not limit the present invention.

Embodiment 1：Obtain the cultivated silkworm peptidoglycan identification gene of Protein S 1

TaKaRa companies are purchased from for the high-fidelity enzyme used in PCR amplification genes, primer is limited by Shanghai JaRa bioengineering Company synthesizes.

First, the nucleotide sequence announced according to GenBank（NM_001043371）Primer is designed, sense primer is 5'- CGCAGGATCCGACATGGCCCGCCTCC -3'（BamH1）, anti-sense primer is 5'- ATTAGCGGCCGCTC

TTGATGGAGTCCACGTTCT -3'(Not1).Secondly, fractionation of fatty body extracts mRNA from silkworm, and reverse transcription is cDNA.Using cDNA as template, performing PCR is entered by designed primer, the cultivated silkworm peptidoglycan identification gene of Protein S 1 is amplified.With 1% Ago-Gel is detected（Referring to Fig. 1）.

Embodiment 2：Cultivated silkworm peptidoglycan recognizes the structure of the gene expression bacterium of Protein S 1

Host Strains DH5 α and BL21 for structure（DE3）, PET-30a plasmids preserve by this laboratory, DH5 α and BL21（DE3） Competence bacterium is made.BamH1 and Not1, ligase is purchased from TaKaRa companies.Plasmid extraction kit is public purchased from OMAGA Department.

Double digestion is carried out to the peptidoglycan recognition protein S1 genes and PET-30a of amplification, using quick ligase by double enzymes Cut peptidoglycan recognition protein S1 genes to be connected on the PET-30a of same double digestion, in 16 DEG C of water-bath 2h, and convert DH5 α impressions State bacterium.The DH5 α of conversion are coated on containing kanamycins（30μg/mL）LB solid mediums on, 37 DEG C of inversions were cultivated Night.Bacterium colony on flat board is subjected to bacterium solution PCR checkings, verified using two pairs of primers（Referring to Fig. 2）.Bacterium solution PCR is verified Correct bacterium colony incubated overnight, extracts plasmid with plasmid extraction kit and is verified for double digestion（Referring to Fig. 3）, checking is correct Purpose cloning and sequencing.Successful purpose cloned plasmids conversion BL21 is sequenced（DE3）, filter out the bacterium colony containing target gene.

Embodiment 3：Induce the expression of destination protein

By the BL21 containing target gene（DE3）Bacterium colony is chosen in containing kanamycins（30μg/mL）LB fluid nutrient mediums in, 37 DEG C Overnight incubation, then by 1:100 dilutions, which are inoculated in, new contains kanamycins（30μg/mL）LB fluid nutrient mediums in 37 DEG C culture To OD values about 0.5 or so, final concentration of 0.8mM IPTG and 20% glucose induction is added, induction time is 5h, 4 DEG C 4500rpm centrifuges 25min, collects thalline, carries out 12%SDS-PAGE and verifies whether expression（Referring to Fig. 4）.

Embodiment 4：Purify destination protein

Nickel ion chelating magnetic bead is purchased from castor nanosecond science and technology（Suzhou）Co., Ltd.

By the bacterium of collection with 20mL1 × PBS twice, 4 DEG C of 4500rpm centrifuge 25min, collect thalline.With Thalline is resuspended in 10mL1 × PBS, ultrasonic on ice（Power：25%, operation 3 seconds, interval 8 seconds, 25 times）Cracking, 4 DEG C of 12000rpm 10min is centrifuged, takes supernatant precipitation to carry out 12%SDS-PAGE respectively, to determine the solubility of destination protein（Referring to Fig. 5）.

Sample treatment：Precipitation 10mL inclusion body cleaning solutions（1M guanidine hydrochlorides, 20mM sodium phosphates, 500mMNaCl, PH=7.8） It is resuspended, supernatant is abandoned in centrifugation, is repeated once.Precipitation 10mL inclusion body lysates（6M guanidine hydrochlorides, 20mM sodium phosphates, 500mMNaCl,PH=7.8）It is resuspended, centrifugation obtains supernatant.

Magnetic bead is pre-processed：Magnetic bead is fully mixed, 1mL suspension containing magnetic beads is drawn in centrifuge tube, centrifuge tube is placed in magnetic On separator, preservation liquid is removed after solution is clarified.Centrifuge tube is removed from magnetic separator.Add 10mLBufferA（8M urinates Element, 20mM sodium phosphates, 500mMNaCl, PH=7.8）Magnetic bead is suspended again, centrifuge tube is placed on magnetic separator, magnetic point From removing supernatant, repeated washing 2 times is with balance nickel ion chelating magnetic bead.

Magnetic bead is combined with destination protein：Added in magnetic bead in sample handling procedure and centrifuge obtained supernatant, by centrifuge tube It is placed on rotary mixer and rotates 20min, destination protein is combined with magnetic bead.Centrifuge tube is placed on magnetic separator afterwards, Magnetic Isolation, removes supernatant.

Magnetic bead is washed：Sequentially add 10mLBufferB（8M urea, 20mM sodium phosphates, 500mMNaCl, PH=6.0）With BufferC（8M urea, 20mM sodium phosphates, 500mMNaCl, PH=5.3）Magnetic bead is washed, with except foreigh protein removing.Finally plus Entering 10mLBufferC makes magnetic bead suspend again, suspension containing magnetic beads is transferred in new centrifuge tube, to prevent the pollution of foreign protein.

Destination protein is eluted：Use 5mLBufferD（8M urea, 20mM sodium phosphates, 500mMNaCl, PH=4.0）Elute purpose Albumen.

Destination protein renaturation：Using dialysis to inclusion body carry out renaturation, extracellular fluid dialysis be respectively A (20mM sodium phosphates, 500mMNaCl, 4M urea, pH 4.0), B (20mM sodium phosphates, 500mMNaCl, 2M urea, pH 4.0), C (20mM phosphoric acid Sodium, 500mMNaCl, 1M urea, pH 4.0), D (20mM sodium phosphates, 500mMNaCl, 0M urea, pH 4.0), every time dialysis Time is 4 hours.

Embodiment 5：12%SDS-PAGE is analyzed and Western blot checkings

Primary antibody（anti-His）Secondary antibody（Sheep anti-mouse igg）Purchased from Nanjing Vazyme Biotechnology Co., Ltd.

12%SDS-PAGE is analyzed：The destination protein for taking 100 μ L to purify adds 20 μ L 5 × SDS Loading Buffer and boiled 5min, 12000rpm centrifuge 5min, take 20 μ L loadings.12%SDS-PAGE is carried out, 2h is dyed with coomassie brilliant blue staining liquid, is used Coomassie brilliant blue destainer decolourizes to stay overnight（Referring to Fig. 6）.

Western blot are verified：By on the albumen of purifying to PDVF films, 1h is closed with 5% skimmed milk power, is washed with TBST Wash 4 times, add primary antibody（anti-His 1:1000 dilutions）, 4 DEG C overnight, and PDVF films are washed 4 times with TBST, adds secondary antibody（Goat-anti Mouse IgG 1:5000 dilutions）1h, TBST are washed 4 times, add nitrite ion, ECL colour developings（Referring to Fig. 7）.

The condition that above-described embodiment more optimizes for the present invention, provable by Fig. 6 and Fig. 7, the albumen being purified into is preferable.This The experimental method of invention is simultaneously not restricted to the described embodiments, on the premise of experimental principle of the present invention, the modification done and is changed Enter to belong within protection scope of the present invention.

SEQUENCE LISTING

<110>Jiangsu University

<120>A kind of expression of cultivated silkworm peptidoglycan identification albumen and purification process

<160>2

<170> Patent In version 3.3

<210>SEQ ID NO.1

<211>26

<212>DNA

<213>Artificial sequence

<220>

<221>

<222>（1）..（26）

<400>1

CGCAGGATCC GACATGGCCC GCCTCC 26

<110>Jiangsu University

<210>SEQ ID NO.2

<211>33

<212>DNA

<213>Artificial sequence

<220>

<221>

<222>（1）..（33）

<400>2

ATTAGCGGCC GCTCTTGATG GAGTCCACGT TCT 33

Claims (3)

1. the method that cultivated silkworm peptidoglycan recognizes the prokaryotic expression of Protein S 1, it is characterised in that：

The sense primer for designing primer is 5'- CGCAGGATCCGACATGGCCCGCCTCC -3',

Anti-sense primer is 5'- ATTAGCGGCCGCTCTTGATGGAGTCCACGTTCT -3；

Restriction enzyme site is inserted in the primer of design, in order to which cultivated silkworm peptidoglycan is recognized into the gene of Protein S 1 with the method for molecular cloning It is cloned into prokaryotic expression carrier PET-30a, the prokaryotic expression carrier built is converted into DH5 α, bacterium solution PCR and digestion are tested The positive colony sequencing of card, will be sequenced correct positive colony and extracts plasmid conversion BL21（DE3）.

2. the method that cultivated silkworm peptidoglycan recognizes the prokaryotic expression of Protein S 1, it is characterised in that correct BL21 will be sequenced（DE3）Overnight Culture, then takes 40 μ L bacterium solutions to add the new nutrient solutions of 4mL, continues to cultivate to OD values about 0.5 or so, adds final concentration of 0.8mM isopropylthiogalactoside（Abbreviation IPTG）With the glucose induction that mass concentration is 20%, induction time is 5h； Because the albumen of expression carries His labels, it can be purified with nickel ion chelating magnetic bead.

3. the method that nickel ion chelating magnetic bead carries out purifying inclusion body protein, it is characterised in that carry out as steps described below：

（1）The bacterium solution of induction is centrifuged, precipitation is collected, precipitation is washed twice with 1 × PBS, supernatant is abandoned in centrifugation, precipitation adds suitable 1 × PBS of amount simultaneously suspends, ultrasonic on ice（Power：25%, operation 3 seconds, interval 8 seconds, 25 times）Cracking, centrifugation, takes supernatant respectively 12%SDS-PAGE is carried out with precipitation, to determine the solubility of destination protein；

（2）Sample treatment：Precipitate with appropriate inclusion body cleaning solution（1M guanidine hydrochlorides, 20mM sodium phosphates, 500mMNaCl, PH= 7.8）It is resuspended, supernatant is abandoned in centrifugation；

Precipitate with appropriate inclusion body lysate（6M guanidine hydrochlorides, 20mM sodium phosphates, 500mMNaCl, PH=7.8）It is resuspended, centrifuges To supernatant；

（3）Magnetic bead is pre-processed：Use Buffer A（8M urea, 20mM sodium phosphates, 500mMNaCl, PH=7.8）Balance nickel ion chela Magnetic bead magnetic bead is closed to be combined with destination protein：Added in magnetic bead in sample handling procedure and centrifuge obtained supernatant, make destination protein Combined with magnetic bead；

（4）Magnetic bead is washed：Use Buffer B（8M urea, 20mM sodium phosphates, 500mMNaCl, PH=6.0）With Buffer C（8M urinates Element, 20mM sodium phosphates, 500mMNaCl, PH=5.3）Magnetic bead is washed, with except foreigh protein removing；

（5）Destination protein is eluted：Use BufferD（8M urea, 20mM sodium phosphates, 500mMNaCl, PH=4.0）Elute purpose egg In vain；

（6）Destination protein renaturation：Renaturation is carried out to inclusion body using dialysis.