CN106046136A

CN106046136A - Eimeria tenella microneme protein-2 mutant EtMIC2-2119 of chickens

Info

Publication number: CN106046136A
Application number: CN201610390480.6A
Authority: CN
Inventors: 赵孝民; 陈正涛; 韩林臻; 李宏梅; 王方昆
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2016-06-03
Filing date: 2016-06-03
Publication date: 2016-10-26
Anticipated expiration: 2036-06-03
Also published as: CN106046136B

Abstract

The invention relates to the field of gene engineering and provides an eimeria tenella microneme protein-2 mutant EtMIC2-2119 of chickens. The mutant is obtained as follows: in-vitro orthogenesis is performed with EtMIC2 (GenBank:KC333870.1) of wild eimeria tenella SD-01 as basic protein, a gene mutation library is established, yeast is used for surface display of a protein mutant library, altered protein with immunogenicity and immune protection remarkably improved is screened by means of sorting by a flow cytometry, animal immune protection effect evaluation and the like, and the mutant is obtained finally. The mutant altered protein has 11 amino acid mutation sites; the immune protection effect ACI of altered EtMIC2 is improved by about 25.06% while compared with that of natural EtMIC2, and the mutant can serve as a vaccine to be applied in the field of resistance to infection by eimeria tenella of the chickens.

Description

A kind of chicken Eimeria Tenella micro-line egg-2 mutant EtMIC2-2119

Technical field

The present invention relates to genetic engineering and field of genetic engineering, it is provided that a kind of chicken Eimeria Tenella micro-line egg-2 is dashed forward Variant EtMIC2-2119.

Background technology

Chicken coccidiosis is the Intestinal protozoan disease that a class is caused by the entozoic Eimeria of special sexual cell.The most every Year whole world is used for preventing and treat coccidiosis cost can be more than 3,000,000,000 dollars, and China there are about hundred million RMB more than 30 every year.At present The prevention and control of coccidiosis rely primarily on medicine and vaccine, and the appearance along with drug-resistant worm plant and the legislation for coccidiostat limit, People more pay attention to immunoprophylaxis.Vaccine on market is mainly the mixing of multiple chicken Eimeria egg capsule alive, has very well Immune protective effect, but owing to live vaccine exists safety problem and higher production cost, researchers will be more Energy is for the development of the novel molecular vaccine based on subunit.

Novel molecular vaccine development relies on has preferable antigenicity and immunogenic antigen, due to complicated the resisting of coccidiosis Original structure and life cycle, the restriction of the difficulty of Antigen Identification and antigen immunogenicity screening hinders subunit vaccine development Progress.Researchers have also carried out substantial amounts of research to improve the immune protective effect of antigen, including the development of new adjuvant, benefit The nonspecific immunity etc. of raw bacterium induction, but these all can not fundamentally improve the immunogenicity of antigen.Therefore, how to obtain Obtain a kind of efficient novel vaccine and become one of problem demanding prompt solution.

Summary of the invention

For problems of the prior art, the invention provides a kind of chicken Eimeria Tenella micro-line egg-2 sudden change Body EtMIC2-2119 and encoding gene thereof, this mutant is with the EtMIC2 of wild type Eimeria tenella SD-01 (GenBank:KC333870.1) based on, protein carries out lactam enzyme by directional anagenesis in vitro, and sets up gene mutation storehouse, uses yeast table Face display protein mutation library, applies the means such as selected by flow cytometry apoptosis and animal immune protected effect evaluation, screens immunogen The variant protein matter that property and immune protective efficiency significantly improve finally obtains, and this mutant variation protein has 11 aminoacid Mutational site.EtMIC2-2119 more natural EtMIC2 immune protective effect ACI after variation improves about 25.06%, can conduct Vaccine is applied in the fields such as opposing chicken eimeria tenella infection.

The dominant mechanism that inventor applies in the present invention is as follows:

Orthogenesis is widely used in protein engineering field, and one of the most important application improves the immunity of antigen exactly Originality.Change immunoreation and the Immune discrimination for antigen of key amino acid have a significant impact, such as reversible inhibition host Immunoreation, affect the activation of T cell, improve protein immunogenic etc., use random mutation to build gene mutation storehouse to be One of method that orthogenesis is conventional.

And EtMIC family protein has important effect in terms of dividing a word with a hyphen at the end of a line coccidiosis, invading host cell and intracellular existence, EtMIC2 protein all can be expressed in the whole biocycle of Eimeria tenella, research show EtMIC2 gene or Protein can react by inducing moiety immunoprotection, is good vaccine candidate antigen.

Therefore the present inventor uses random mutation technology to be oriented evolution for EtMIC2, sets up gene mutation Storehouse, uses yeast surface display technology to combine FACS and animal immune protection test screening purpose variant protein matter, thus changes The immunogenicity of kind EtMIC2 protein, provides technical support for opposing chicken eimeria tenella infection.

The concrete technical scheme of the present invention is as follows:

The EtMIC2 nucleotide sequence (GenBank:KC333870.1) that first present invention announces according to GenBank manually closes Becoming natural EtMIC2 sequence, its nucleotide sequence is as shown in Seq ID No:3, by this EtMIC2 nucleotide sequence and pEASY-T1 Carrier connects modeling EtMIC2-T1.

Inventor utilizes fallibility PCR expand above-mentioned EtMIC2-T1 and obtain mutant, sets up gene mutation storehouse afterwards, makes With yeast surface display protein mutation storehouse, then the application hands such as selected by flow cytometry apoptosis and animal immune protected effect evaluation Section, the variant protein matter that screening immunogenicity and immune protective efficiency significantly improve, it is finally obtained sudden change of the present invention Body, named EtMIC2-2119, this mutant nucleotide sequence is as shown in Seq ID No:1, and the aminoacid sequence of its coding is such as Shown in Seq ID No:2, by comparing with unmutated EtMIC2, this mutant variation protein has 11 amino acid mutation positions Point, concrete mutated site is as follows:

Different amino acids and site thereof compared with EtMIC2-2119 with EtMIC2

Inventor, after obtaining said mutation body, has carried out immune protective effect checking to it, found that compared to Unmutated EtMIC2 protein, mutant EtMIC2-2119 Western Immuno group body weight increases notable；Mutant EtMIC2- The caecum average lesion score of 2119 Western Immuno groups is significantly lower than unmutated EtMIC2 immune group；Mutant EtMIC2- 2119 Western Immuno groups in terms of gram feces egg sac number compared with native protein EtMIC2, though Oocyst Production of Three has reduced, But do not show notable difference；The ACI of mutant EtMIC2-2119 Western Immuno group is 172.48, and ratio is unmutated EtMIC2 protein immunization group is high by about 25.06%, has good anticoccidial effect, it is seen that mutant provided by the present invention EtMIC2-2119 can apply in the fields such as opposing chicken eimeria tenella infection as vaccine.

In sum, present invention obtains a kind of brand-new chicken Eimeria Tenella micro-line egg-2 mutant EtMIC2- 2119, this mutant is about 25.06% than improving for mutant immune protective effect ACI, can be soft opposing chicken as vaccine The fields such as tender eimeria tenella infection are applied.

Accompanying drawing explanation

Fig. 1 is yeast surface display protein EtMIC2 and sudden change EtMIC2 protein S DS-PAGE testing result gray scale Figure,

In figure, M is Marker；1 is EBY100 negative control；2 is EtMIC2 protein；3 is EtMIC2-2119 variation egg White matter；

Fig. 2 is yeast surface display protein EtMIC2 and sudden change EtMIC2 protein Western blot testing result Gray-scale map,

In figure, M is Marker；1 is EtMIC2 protein；2 is EtMIC2-2119 variant protein matter；

Fig. 3 is that selected by flow cytometry apoptosis shows the yeast cells schematic diagram having EtMIC2 albumen.

Detailed description of the invention

Test material and reagent

1, bacterial strain, carrier: various bacterial strains involved in the present invention are conventional bacterial strain, can the most directly reach；Institute The cloning vehicle pEASY-T1 used is purchased from Quan Shi King Company；Expression vector pET-30a (+), use this area routine techniques to obtain Obtain or buy.

2, main agents and medicine: rTaq enzyme, dNTPs, DNA Marker, Protein Marker, PCR primer purification try Agent box, DNA glue reclaim test kit and plasmid extraction kit is purchased from Beijing Quanshijin Biotechnology Co., Ltd；Restriction enzyme Enzyme BamH I, Nhe I, Hind III and Sal I are purchased from Dalian treasured biological engineering company limited；T4DNA ligase is purchased from Fermentas；FITC labelling sheep anti-mouse igg, HRP labelling sheep anti-mouse igg and IgA are purchased from Beijing limited public affairs of Bo Aosen biotechnology Department；Quick Start^TMBradford 1x Dye Reagent is purchased from Bio-Rad company；His·Purificat Ion Kit is purchased from Merck company；Complete Freund's adjuvant and incomplete Freund's adjuvant etc. are purchased from Sigma company；Dithiothreitol, DTT (DTT) purchased from Sigma company；Yeast plasmid rapid extraction test kit is purchased from the green skies, Shanghai Bioisystech Co., Ltd, other Conventional reagent is domestic analytical pure.

3, the preparation of culture medium:

(1) LB fluid medium: use electronic balance to weigh yeast extract 5g, tryptone 10g, sodium chloride 10g, molten Solution in 1L deionized water, 121 DEG C of autoclaving 20min.

(2) solid LB media: preparation LB fluid medium 100mL by the above process, addition 1.5g agar powder, 121 DEG C Autoclaving 20min, pours in culture dish after cooling.

(3) solid LB/AMP (Kan) culture medium: preparation LB fluid medium 100mL by the above process, treats that temperature is down to not When scalding one's hand, add 100 μ L Amp (Kan) stock solutions, after mixing, pour in culture dish, cool down rear 4 DEG C of preservations.

(4) YPD fluid medium: use electronic balance to weigh yeast extract 10g, tryptone 20g, glucose 20g It is dissolved in 1L deionized water, 121 DEG C of sterilizing 15min.

(5) solid YPD culture medium: preparation YPD fluid medium 100mL by the above process, addition 1.5g agar powder, 121 DEG C autoclaving 20min, pours in culture dish after cooling.

(6) SD-CAA fluid medium: use electronic balance to weigh yeast nitrogen 6.7g, acid hydrolysis casein 5g, glucose 20g is dissolved in 1L deionized water, uses magnetic stirring apparatus fully to dissolve, 121 DEG C of autoclaving 15min.

(7) solid SD-CAA culture medium: will add 1.5-2g agar powder in 100ml SD-CAA fluid medium, high pressure goes out After bacterium, it is poured into culture dish.

(8) SG-CAA fluid medium: use electronic balance to weigh without aminoacid without ammonium sulfate yeast nitrogen 6.7g, sour water Solve casein 5g to be dissolved in 1L deionized water, use magnetic stirring apparatus fully to dissolve, 121 DEG C of autoclaving 15min, add afterwards The galactose of the filtration sterilization of 20g 4 DEG C preservation.

4, the present embodiment does not does the experimental methods of molecular biology illustrated in detail, all with reference to " Molecular Cloning: A Laboratory Guide " concrete grammar listed in (third edition) J. Pehanorm Brooker one book carries out, or according to test kit and product description Carry out.

The acquisition of embodiment 1 mutant EtMIC2-2119

The EtMIC2 nucleotide sequence (GenBank:KC333870.1) that first basic invention announces according to GenBank is artificial Synthesis of natural EtMIC2 sequence, the EtMIC2 nucleotide sequence obtained, its nucleotide sequence as shown in Seq ID No:3, it After it is connected with pEASY-T1 carrier modeling EtMIC2-T1；With EtMIC2-T1 as template, use EtMIC2-951-F and EtMIC2-951-R is primer, utilizes Divers ify PCR Random Mutagenes is Kit (Clontech, USA) to enter Row fallibility PCR amplification EtMIC2 mutant obtains fallibility PCR primer；Wherein the primer is as shown in table 1:

Mutant EtMIC2-2119 specific primer described in table 1.

The preparation of mutant described in embodiment 2

By in embodiment 1, the fallibility PCR primer of acquisition is in the case of unpurified, after being connected with pEASY-T1 carrier, turn Enter Top 10 competent cell, be coated with ammonia benzyl LB agar plate, cultivate until monoclonal bacterium colony increases to 10 in 37 DEG C of incubators⁷For Only, EtMIC2 gene mutation storehouse is built；

The above-mentioned recombiant plasmid including gene mutation library and pCTCON2 plasmid, use Nhe I and Sal I to carry out Double digestion, transfects saccharomyces cerevisiae EBY100 and (is purchased from after being connected with pCTCON2 carrier in the gene mutation storehouse of double digestion Invitrogen), obtain recombinant bacterial strain EBY100/EtMIC2, PCR and identify positive bacterium colony (design the primer is as shown in table 2)； Take the bacterial strain EBY100 containing recombiant plasmid, on coating SD-CAA solid medium in 30 DEG C of incubators, cultivate 48h, until yeast Bacterium bacterium colony is 10⁸Till individual, build EtMIC2 mutein storehouse recombinant bacterial strain EBY100/EtMIC2；

Table 2

The ratio taking the EBY100 bacterial strain 1:100 by volume containing recombiant plasmid is inoculated in the SD-CAA of 100mL, and 30 DEG C, 220rpm cultivates about 8h, stops cultivating to OD600 about about 1.0, by thalline 4 DEG C under sterile conditions, and 3000r/min, Centrifugal 5min, discards supernatant, use sterile distilled water wash three times thoroughly to remove glucose, 3000r/min, centrifugal 5min, Using the freshly prepared resuspended thalline of SG-CAA culture medium 100mL, and be transferred in the triangular flask of sterilizing, 30 DEG C, 220rpm lures Lead and cultivate about 12h；

The yeast concentration of abduction delivering is adjusted to 5x10⁷Individual/10 μ L, add the DTT solution of final concentration of 100mM, and 4 DEG C effect overnight, period constantly shakes.The protein solution of acquisition is joined in bag filter, uses PBS molten under the conditions of 4 DEG C Liquid dialysis repeatedly (each 2h) removes DTT, 10,000rpm, centrifugal 1min, draws supernatant, uses SDS-PAGE and Western Blot analyzes expression, and result shows positive band (as illustrated in fig. 1 and 2) occur at about 50kDa；

Taking the yeast cell of above-mentioned abduction delivering, the PBS-BSA (1mg/mL) using pH to be 7.2 washes 2 times, and 10,000r/ Min, centrifugal 1min, be resuspended in thalline in the rabbit source anti-EtMIC2 multi-resistance (utilizing prior art to prepare) of 40 μ L1:200 dilutions, Mixing, ice bath 30min, the PBS-BSA (1mg/mL) using pH to be 7.2 wash 2 times, and 10,000r/min, centrifugal 1min, by thalline Being resuspended in the FITC labelling mouse-anti rabbit igg monoclonal antibody of 50 μ L1:75 dilutions, mixing, ice bath 30min, using pH is the PBS-BSA of 7.2 (1mg/mL) 2 times are washed, 10,000r/min, centrifugal 1min, BD FACSAria Cel l-Sort ing System carries out aseptic Screening is containing mutants which had, to show the different of unmutated EtMIC2 protein EtMIC2 anti-from rabbit source polyvalent antibody affinity For comparison, the delineation more weak door P2 of fluorescence signal (accounting for the 2.7% of total cell number, its fluorescence intensity is: 2757.18, represent with EtMIC2 multi-resistance affinity is more weak) (as shown in Figure 3), after three step sizing, coating SD-CAA solid agar flat board, 30 DEG C cultivate 48h, collect yeast list bacterium colony 10 strain.

Routine techniques extracts the above-mentioned 8 saccharomycete pCTCON2 recombiant plasmid containing mutant gene, with this plasmid as mould Plate, with His-EtMIC2-F and His-EtMIC2-R shown in table 2 as primer, uses PrimeSTAR DNA Polymerase (Takara) carry out PCR and expand EtMIC2 mutant gene, by above-mentioned PCR primer and pET30a, use BamH I and Hind III carries out double digestion, converts BL21 bacterial strain and obtain recombinant bacterial strain BL21/EtMIC2-1130 after connection.Positive colony presses 1:100's Ratio is inoculated in the LB culture medium containing kalamycin resistance, at 37 DEG C, cultivates 3h, extremely in the constant-temperature table of 220rpm During OD600 ≈ 0.6, adding the IPTG solution abduction delivering 4h of final concentration of 1mM in each pipe, 8,000rpm are centrifuged 5min, receive Collection thalline, 1 × combine buffer solution thalline 2 times, use with the addition 4mL 1 × combination of every 100mL culture medium gained thalline slow Rush the resuspended thalline of liquid proportional.After ultrasonic disruption (power 300W, opens 3sec, stops 6sec), 16,000g are centrifuged 20min collects Supernatant and precipitation, precipitation adds 5mL buffer ratio by every 100mL culture, adds 1 × combination buffering of final concentration of 8M carbamide Liquid, thoroughly dissolves inclusion body, and 16,000g are centrifuged 30 minutes and again collect supernatant, collect supernatant mixing for twice, and adjusting pH is 8.0, makes Use 0.45um membrane filtration, for His Bind protein purification (according to HisPurificat ion Kit explanation Book is carried out), it is thus achieved that mutant protein, named EtMIC2-2119, its nucleotide sequence is as shown in Seq ID No:1, and it encodes Aminoacid sequence as shown in Seq ID No:2, by comparing with unmutated EtMIC2, this mutein has 11 amino Acid mutation site.

According to Quick Start^TMBradford 1x Dye Reagent description, use 0.125,0.25,0.5, 0.75, each 5 μ L of the BSA standard substance of 1.0,1.5 and 2.0mg/mL join in the EP pipe containing 250 μ L dye reagents, the most mixed Even, room temperature effect 5min, each gradient does three repetitions；Take each 5 μ L of testing protein quality sample to join containing 250 μ L dyestuff examinations In the EP pipe of agent, fully mixing, room temperature effect 5min, each gradient does three repetitions, and mixed liquor joins the hole of ELISA Plate In, use microplate reader to measure OD595；Drawing standard curve, calculating mutant protein concentration according to the formula drawn is 1.36mg/ mL。

Embodiment 3 mutant EtMIC2-2119 immune protective effect judges

1 Japanese instar chickling is weighed, and rejects the chicken that bodyweight difference is bigger.Altogether 100 chickling are divided into 4 groups, respectively PBS-I (the most immune not infected group), PBS-II (the most immune infected group), native protein EtMIC2, mutant EtMIC2 immune group, often group 25, raise in isolation cover.When 7 age in days, each group chicken carries out subcutaneous multiple spot immunity Freund's complete adjuvant emulsifying respectively The protein of final concentration of 0.5mg/mL or PBS 100 μ L, carry out booster immunization incomplete Freund's adjuvant emulsifying when 14 age in days The protein of the final concentration of 0.5mg/mL of correspondence or PBS 100 μ L.21 Japanese instar chicklings are equal oral vaccination 30 in addition to PBS-I group, 000 Sporulated Oocysts.According to immunity chicken body weight growth pattern, cecal lesion score, gram feces egg sac number (OPG), anti-ball The index judgement mutant protein immune protective effects such as worm index (ACI):

One, body weight growth pattern measures: concrete grammar is as follows:

Carry out weighing by plumage when 21 ages in days (before attacking worm) and 31 ages in days (after attacking worm), record weighing results, and calculate flat All weightening finish (average weight gain (g)=31 age in days average weight (g)-21 age in days average weight (g)) and the relative weight gain amount (the relative weight gain Rate (%)=(weightening finish of the weightening finish of infected group/do not infect not immune group) × 100%).Result shows: compared with PBS-II group, Immunity native protein EtMIC2 group and mutant EtMIC2-2119 group body weight increase notable, compared to unmutated EtMIC2 egg White matter group, mutant EtMIC2-2119 group body weight increases notable.

Two, cecal lesion score measures: concrete grammar is as follows:

According to (1970) 5 points of principles processed of Johnson and Reid, 5d after infecting coccidiosis, often group is cutd open and is killed 6 chickens, takes caecum Look first at outward appearance petechia, swelling situation, then longitudinally cut caecum, observed content thing state open, use tap water to dispose Content observes the bleeding of caecum inwall, and comprehensively these pathological changes carry out evaluation and score.Result shows: compared with PBS-II group The score of EtMIC2, EtMIC2-2119 immune group caecum average lesion significantly reduces, and the caecum of mutant EtMIC2-2119 group Average lesion score is significantly lower than unmutated EtMIC2 immune group.

Three, gram feces egg sac number (OPG): concrete grammar is as follows:

After attacking worm, 5d to 10d starts to collect each group of feces every day respectively, mixes rear 4 DEG C of preservations.Often organize and take 1g, add Enter 10mL tap water and carry out 10 times of dilutions, after stirring, use 400 mesh filter screens to grind and be filtered to remove big feces Granule.The filtrate 10 μ L taking fully mixing drips with on microscope slide, and in the 10 μ L diluents that count under low power lens, coccidian oocyst is total, According to formula (OPG=coccidian oocyst sum × 10⁴) calculate OPG and protective rate.Result shows: compared with PBS-II group, albumen Matter EtMIC2, EtMIC2-2119 immune group Oocyst Production of Three all significantly reduces, but variant protein matter EtMIC2-2119 group gram Feces egg sac number aspect is compared with native protein EtMIC2 group, and it is the most notable that egg sac number reduces difference.

Four, anticoccidial index (ACI) is calculated: concrete grammar is as follows:

By Merck company of U.S. formula: ACI=(survival rate+body weight increase rate) × 100-(pathological changes value+egg capsule value).Root The curative effect of anticoccidial is judged according to ACI；ACI 120 is invalid, 120 < ACI 160 be medium effectively, 160 < ACI 180 are Well, ACI > I80 is outstanding.Result shows: the ACI of unmutated EtMIC2 Western Immuno group is 137.92, has medium Anticoccidial protected effect, the ACI of mutant EtMIC2-2119 Western Immuno group is 172.48, than unmutated EtMIC2 albumen Immune group is high by about 25.06%, has good anticoccidial effect.

Claims

1. chicken Eimeria Tenella micro-line egg-2 mutant, it is characterised in that the named EtMIC2-of this mutant 2119, its nucleotide sequence is as shown in Seq ID No:1, and the aminoacid sequence of its coding is as shown in Seq ID No:2.

2. chicken Eimeria Tenella micro-line egg-2 mutant described in claim 1 is resisting chicken tender Amy that ball as vaccine Application on insect infection.