CN103497249B - Preparation of baculovirusHearNPV LEF-9 protein polyclonal antibody - Google Patents

Abstract

The invention relates to preparation of a baculovirusHearNPV LEF-9 protein polyclonal antibody. The preparation comprises: designing a primer and cloning lef-9 gene; constructing recombinant plasmid pET-28a-lef-9 by prokaryotic expression; transforming the constructed recombinant plasmid pET-28a-lef-9 into escherichia coli BL21 for construction of a recombinant bacterial strain capable of expressing LEF-9 protein; performing inducible expression and purification on LEF-9 protein, taking the prepared baculovirusHearNPV LEF-9 protein as an antigen, employing a conventional polyclonal antibody preparation method to prepare antiserum, and purifying antiserum to obtain the polyclonal antibody. The preparation method of the invention helps to realize prokaryotic expression of the baculovirusHearNPV LEF-9 protein, is applicable to preparation of the polyclonal antibody of the baculovirusHearNPV LEF-9 protein in a later period, and establishes a base for further research on functional characteristics of LEF-9 protein. The preparation of the baculovirusHearNPV LEF-9 protein helps people to have more comprehensive and deeper understanding about baculovirus RNA polymerase, and establishes a base for construction of an efficient in-vitro transcription system by utilizing the LEF-9 protein.

Description

The preparation of baculovirus HearNPV LEF-9 protein polyclone antibody

Technical field

The present invention relates to a kind of preparation method of antibody, particularly the preparation method of baculovirus HearNPV LEF-9 protein polyclone antibody.

Background technology

Baculovirus is the specific pathogen of insect, it is monoid maximum in known insect viruses, also be find the earliest, most study and there are the insect viruses of extremely important practical value, baculovirus is subject to the extensive concern of academia as efficient eukaryotic gene expression vector system, effective viral pesticide and potential gene therapy vector system.

Baculovirus Gene group coding several genes, according to the timing of genetic expression, the gene of baculovirus is divided into early gene and the large class of late gene two.Because current most of evidence shows, the temporal expression of virogene be by sequential before the expression product of a papova gene, transcribing of a papova gene directly or indirectly after its sequential of trans-activation, thus regulates and mainly occurs on transcriptional level.Therefore the viral rna polymerase as Retroviral late gene just becomes the key factor regulating early and late transcription gene transcriptional expression.

Baculovirus RNA polymerase is by virus induction and coding, and after viral genome starts to copy, early gene stops transcribing, and viral rna polymerase specific recognition contains the late gene promoter of conserved sequence TAAG and starts transcribing of late gene.Research shows that viral rna polymerase is only made up of 4 conservative genes lef-4, lef-8, lef-9 and p47 of encoding viral, and the complex body that these four kinds of albumen are formed can Retroviral late gene in vitro.Although scientist has just found the existence of this enzyme as far back as the eighties in last century, we are also very limited to its understanding up to now.Existing known in its four ultimate constituents P47 be positioned in nucleus, it is the indispensable gene of late gene transcription, LEF-4 plays mRNA and adds cap and grappling promotor, LEF-8 is relevant with polysaccharase specific recognition late promoter, and infers the catalytic site together constituting RNA polymerase with LEF-9.

LEF-9 is second largest albumen in viral rna polymerase, if the function of HearNPV LEF-9 subunit in viral rna polymerase can be resolved, by make people to baculovirus RNA polymerase have one more comprehensively with deep understanding, build efficient eukaryotic gene expression vector system for utilizing it further, prepare effective viral pesticide and exploitation gene therapy vector system lay the foundation.In order to study this albumen, prepare its polyclonal antibody be one necessary early stage basis.

Summary of the invention

The object of this invention is to provide a kind of method preparing baculovirus HearNPV LEF-9 protein polyclone antibody, to study the functional performance of LEF-9 albumen, make people to baculovirus RNA polymerase have one more comprehensively with deep understanding, build efficient in-vitro transcription system for utilizing it further and lay the foundation.

The technical solution used in the present invention is: the preparation of baculovirus HearNPV LEF-9 protein polyclone antibody, and step is as follows:

1) lef-9the clone of gene: design primer 2 8a55F:5 '-gcg ggatccatgtcgaaaacacgtggctcg-3 ' and 28a55R:5 '-gcg aagctttcatattaaaaacatgtctagcaaatg-3 ', with HearNPV genomic dna for masterplate increases lef-9entire reading frame, PCR reaction conditions is: 94 DEG C of denaturation 5min; 94 DEG C of 30s, 59 DEG C of 30s, 68 DEG C of 30s are a circulation, carry out 30 circulations; Last 68 DEG C extend 7min, obtain lef-9gene PCR product;

2) structure of prokaryotic expression carrier: reclaim test kit with gel and reclaim lef-9gene PCR product, uses hindIII and bamhI double digestion, simultaneously with identical two enzyme double digestion prokaryotic expression carrier pET-28a, is connected endonuclease bamhi by T4 DNA ligase with carrier, construction recombination plasmid pET-28a-lef-9;

3) structure of recombinant strains: by the recombinant plasmid pET-28a-lef-9 transformation of E. coli BL21 built, the recombinant bacterial strain of construction expression LEF-9 albumen;

4) abduction delivering of LEF-9 albumen and purifying: by positive for pET-28a-lef-9 single colony inoculation in 5mL Kana resistance LB substratum, 37 DEG C of shaking culture are spent the night, getting overnight culture is inoculated in 2 pipe 250mL fresh LB by 1% volume, 37 DEG C of shaking culture 3h to OD _600nmbe after 0.6, adding IPTG to final concentration is 0.4mmol/L, cultivates 5h, collected by centrifugation thalline;

5) purifying of LEF-9 albumen: the thalline collected in step 4 is resuspended in 5ml lysis buffer,-70 DEG C of multigelations 3 times, ultrasonic disruption, condition is power 200-300 W, and ultrasonic 3s stops 5s, amount to 20min, centrifugal 15 min of 12000 r/min after becoming clarification to bacteria suspension, repeat 3 times, remove insoluble cell debris, obtain the supernatant liquor containing LEF-9 albumen, this supernatant liquor is passed through Ni ²⁺affinity column, then the 250 mM imidazole concentration elution buffer wash-outs using 5 times of column volumes, the albumen that collection elutes also surveys A280, starting to collect sample, until A280 returns to reference line again, obtaining the LEF-9 albumen of purifying when there being protein peak;

6) polyclonal antibody preparation: the LEF-9 albumen obtained with step 5) is immunogen, 1:1 mixes with Freund's complete adjuvant in mass ratio, the conveniently preparation method of polyclonal antibody, immunity 4 rabbit altogether, collect serum, be separated, after purifying, obtain baculovirus HearNPV LEF-9 protein polyclone antibody.

The baculovirus HearNPV LEF-9 protein polyclone antibody of above-mentioned acquisition may be used for the detection of LEF-9 albumen.

The invention has the beneficial effects as follows: by method of the present invention, prepared the polyclonal antibody of LEF-9 albumen, for the functional performance studying LEF-9 albumen is further laid a good foundation.

Accompanying drawing explanation

Fig. 1 is the design of graphics of recombinant plasmid pET-28a-lef-9.

Fig. 2 A is lef-9the SDS-PAGE electrophorogram of gene amplification result.

Wherein, M: molecular criteria; 1: lef-9 gene amplification fragment.

Fig. 2 B is the SDS-PAGE electrophorogram of pET-28a-lef-9 qualification.

Wherein, M: molecular criteria; 1:pET-28a-lef-9 enzyme cuts result.

Fig. 3 is purifying LEF-9 Protein Detection figure.In figure, M: molecular criteria; 1: the LEF-9 albumen of purifying.

Fig. 4 is the western blot detection figure of LEF-9 albumen.Wherein, M:marker; 1: viral LEF-9 albumen.

Embodiment

the preparation of embodiment 1 baculovirus HearNPV LEF-9 protein polyclone antibody

Concrete operation method is as follows:

1. lef-9the clone of gene

Design primer 2 8a55F:5 '-gcg ggatcc(underscore represents atgtcgaaaacacgtggctcg-3 ' bamhI site) and 28a55R:5 '-gcg aagctt(underscore represents tcatattaaaaacatgtctagcaaatg-3 ' hindIII site), with HearNPV genomic dna for masterplate increases lef-9entire reading frame, PCR reaction conditions is: 94 DEG C of denaturation 5min; 94 DEG C of 30s, 59 DEG C of 30s, 68 DEG C of 30s are a circulation, carry out 30 circulations; Last 68 DEG C extend 7min, obtain lef-9 gene PCR product.Reaction product 1% agarose gel electrophoresis is identified, result display obtains 1578bp fragment (Fig. 2 A).

2. the structure of prokaryotic expression expression vector

Reclaim test kit with gel and reclaim purifying lef-9 gene PCR product, use hindIII and bamhI double digestion, simultaneously with identical two enzyme double digestion prokaryotic expression carrier pET-28a, endonuclease bamhi is connected construction recombination plasmid pET-28a-lef-9(Fig. 1 with carrier by T4 DNA ligase), transformation of E. coli DH5 α, kalamycin resistance screening positive strain, extracts plasmid, enzyme cuts qualification positive colony (Fig. 2 B), the raw work order-checking through Shanghai, gene order result is for shown in sequence table SEQ ID NO.1, and order-checking qualification clone is without sudden change.

3. the structure of recombinant strains

by the recombinant plasmid pET-28a-lef-9 transformation of E. coli BL21 successfully constructed, the recombinant bacterial strain of construction expression LEF-9 albumen.

4. the abduction delivering of LEF-9 albumen

By positive for pET-28a-lef-9 single colony inoculation in 5mL LB substratum (Kana 50ug/mL), 37 DEG C of shaking culture are spent the night.Getting overnight culture is inoculated in 2 pipe 250mL fresh LB (Kana 50ug/mL) by 1% volume, 37 DEG C of shaking culture 3h to OD _600nmbe after 0.6, adding IPTG to final concentration is 0.4mmol/L, cultivates 5h, collected by centrifugation thalline.

5. the purifying of LEF-9 albumen:

The thalline collected in step 4 is resuspended in 5ml lysis buffer,-70 DEG C of multigelations 3 times, ultrasonic disruption, condition is power 200-300 W, and ultrasonic 3s stops 5s, amount to 20min, centrifugal 15 min of 12000 r/min after becoming clarification to bacteria suspension, repeat 3 times, remove insoluble cell debris, obtain the supernatant liquor containing LEF-9 albumen, this supernatant liquor is passed through Ni ²⁺affinity column, then the 250 mM imidazole concentration elution buffer wash-outs using 5 times of column volumes, the albumen that collection elutes also surveys A280, starts to collect sample, until A280 returns to reference line again when there being protein peak.The albumen collected is detected by SDS-PAGE.Obtain the LEF-9 albumen of purifying.

6. the preparation of baculovirus HearNPV LEF-9 protein polyclone antibody

With the LEF-9 albumen of the purifying obtained in step 5 for immunogen, immunity is carried out to rabbit: by the LEF-9 albumen of 400mg in mass ratio 1:1 mix with Freund's complete adjuvant, carry out subcutaneous injection under skin around rabbit both shoulders and carry out intramuscular injection at rear thigh, the immunogen of 1/4 is approximately used in each region.Adopt back multi-point injection method after two weeks, namely select 4-6 point subcutaneous injection immunogen in rabbit backbone both sides, amount to 400mg LEF-9 albumen; Interval selects difference injection to carry out three immunity and four immunity respectively at above-mentioned position after 2 weeks and 4 weeks again; Last injection got blood after 2 weeks, collected serum, was separated, after purifying, obtained baculovirus HearNPV LEF-9 protein polyclone antibody.

7. the detection of baculovirus HearNPV LEF-9 protein polyclone antibody

Sf-9 cell is infected with baculovirus HearNPV G4 strain, 72 h before harvest cells infecteds, this sample is detected by western blot with the baculovirus HearNPV LEF-9 protein polyclone antibody of preparation, as shown in Figure 4, result proves that the baculovirus HearNPV LEF-9 protein polyclone antibody of preparation can detect the LEF-9 albumen of virus to result.

<110> Liaoning University

The preparation of <120> baculovirus HearNPV LEF-9 protein polyclone antibody

<160> 3

 

<210> 1

<211> 1560

<212> DNA

<213> lef-9

<400> 1

 

atgtcgaaaa cacgtggctc gattaaacgt aaattgttat gtgatcatac tgaaaaaacg tgcagcaaac gtgtgaaaag caaaattcaa tttgtaacaa aagaaccggt acaattttca ttgctcaccg atcccaatca aataaacaat gtcctcttca taaacataca caattttaaa gtgtttctca agaatttaat tgccgattta aaaaaaataa aaattaattt ttacaacagc ttgttggagc agctgatctc tgtgtactcg gactgcggtc atagaaacga gcacacaaac ttgctgagtc gaatcttggt agccaccagc gtagtcatca ctgatctacc ctcgaacgtt tttttgaaaa aactcaaaac taaccgtttc accgacaata tagactactt gattttaccg aactttgtgc tatgggatca caatttcatt atattcatga acaaagcatt taattcaaaa cacgacaatg gtctgatcga catatcgggc tcgctgcaaa aaatcaaatt aacccacggc gtaattaaag atcaactaca gagcaaaaac ggctatgccg gtcagttttt gtattcgaca ttcttgaata cggcatcgtt ctatgccaac gtgcaatgtt taaacggagc aaacgaaatt gtaccaccga aggccagtct gcgacgctat tatggacgcg atgtgaaaaa tgtacgcgcc tggacaacgc gtcatccgaa catatctcaa ttaagcacac agatatcaag cgtgcgcgaa ccggacaatt acaccgattg gaatgttaaa gtcggcttag gcacgtttac tggcgctaat cgcgactgcg acggtgacaa agaagttatt acttttttgc ctcaacccaa ttcattgata gacttggaat gtctcatgta cggagatccg cgttacaatt tcatttgttt cgacaagaac cgtttatcgt ttgtgtcgca gcaaatatat tatctgcaca aaaacaaaaa acgtatcgaa aaactattgc acagtatgcc tattttatat acactatgga agagctacaa acgttacagc tccatcaatt tggcgacaaa aattgattgg ttgttacgcg attgtgctct cttgctcagc tccaatacca gttttctgct ctacaacaaa ttggctacaa ttatagacaa tgaagaaatg acttgcggcg acgaggaaat atttaatttg gcaggacaat tcaacgacgt catcgaatgc ggagccaaag gcagcgctga tttggtagcg agtactaaaa aatatcgcaa cactcattcc gacgatatag atacaatcgc caagcgtgcc attaccggtt tgaacagcca tatcacgtca cacaatcgag tgaaaatcgg cggtggtgat atctaccaca atacgacagt attgcaaaat gtctatctaa agaacgatta catttgttat aaaaatgaca cgcgtcgtat ttcaagcgtg tgcgcgctgc cgtcgaaatt cctatttcct gaacatttgc tagacatgtt tttaatatga

 

<210> 2

<211> 30

<212> DNA

<213> primer 2 8a55F

<400> 2

 

5’-gcg ggatccatgtcgaaaacacgtggctcg-3’

 

<210> 3

<211> 36

<212> DNA

<213> primer 2 8a55R

<400> 3

 

5’-gcg aagctttcatattaaaaacatgtctagcaaatg-3’

Claims (1)

1. the preparation of baculovirus HearNPV LEF-9 protein polyclone antibody, is characterized in that step is as follows:

1) lef-9the clone of gene: design primer 2 8a55F:5 '-gcg ggatccatgtcgaaaacacgtggctcg-3 ' and 28a55R:5 '-gcg aagctttcatattaaaaacatgtctagcaaatg-3 ', with HearNPV genomic dna for masterplate increases lef-9entire reading frame, PCR reaction conditions is: 94 DEG C of denaturation 5min; 94 DEG C of 30s, 59 DEG C of 30s, 68 DEG C of 30s are a circulation, carry out 30 circulations; Last 68 DEG C extend 7min, obtain lef-9gene PCR product;

2) structure of prokaryotic expression carrier: reclaim test kit with gel and reclaim lef-9gene PCR product, uses hindIII and bamhI double digestion, simultaneously with identical two enzyme double digestion prokaryotic expression carrier pET-28a, is connected endonuclease bamhi by T4 DNA ligase with carrier, construction recombination plasmid pET-28a-lef-9;

3) structure of recombinant strains: by the recombinant plasmid pET-28a-lef-9 transformation of E. coli BL21 built, the recombinant bacterial strain of construction expression LEF-9 albumen;

4) abduction delivering of LEF-9 albumen: by positive for pET-28a-lef-9 single colony inoculation in 5mL Kana resistance LB substratum, 37 DEG C of shaking culture are spent the night, getting overnight culture is inoculated in 2 pipe 250mL fresh LB by 1% volume, 37 DEG C of shaking culture 3h to OD _600nmbe after 0.6, adding IPTG to final concentration is 0.4mmol/L, cultivates 5h, collected by centrifugation thalline;

5) purifying of LEF-9 albumen: the thalline collected in step 4) is resuspended in 5ml lysis buffer,-70 DEG C of multigelations 3 times, ultrasonic disruption, condition is power 200-300 W, and ultrasonic 3s stops 5s, amount to 20min, centrifugal 15 min of 12000 r/min after becoming clarification to bacteria suspension, repeat 3 times, remove insoluble cell debris, obtain the supernatant liquor containing LEF-9 albumen, this supernatant liquor is passed through Ni ²⁺affinity column, then the 250 mM imidazole concentration elution buffer wash-outs using 5 times of column volumes, the albumen that collection elutes also surveys A280, starting to collect sample, until A280 returns to reference line again, obtaining the LEF-9 albumen of purifying when there being protein peak;

6) polyclonal antibody preparation: the LEF-9 albumen obtained with step 5) is immunogen, 1:1 mixes with Freund's complete adjuvant in mass ratio, the conveniently preparation method of polyclonal antibody, immunity 4 rabbit altogether, collect serum, be separated, after purifying, obtain baculovirus HearNPV LEF-9 protein polyclone antibody.