CN109096389B

CN109096389B - Soft-shelled turtle systemic sepsis spherical virus STSSV blocking agent and preparation method and application thereof

Info

Publication number: CN109096389B
Application number: CN201710513244.3A
Authority: CN
Inventors: 陈梅娟; 李登峰
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2017-06-21
Filing date: 2017-06-21
Publication date: 2022-09-23
Anticipated expiration: 2037-06-21
Also published as: CN109096389A

Abstract

The invention relates to a recombinant turtle apolipoprotein B, which is expressed and purified in escherichia coli genetic engineering bacteria Rosetta and has the molecular weight of 48 kDa. The recombinant turtle apolipoprotein B can block the turtle systemic septicemia spherical virus STSSV from infecting turtle cells, can be used as an STSSV blocking agent, and is used for developing antiviral drugs, feed additives and the like.

Description

Soft-shelled turtle systemic sepsis spherical virus STSSV blocking agent and preparation method and application thereof

Technical Field

The invention belongs to the field of gene engineering and aquatic animal disease prevention and control, and particularly relates to a blocking agent of a turtle systemic septicemia spherical virus STSSV, wherein the blocking agent is a recombinant turtle apolipoprotein B.

Background

The turtle is extremely rich in nutrient components, is treasure for the whole body, is a precious economic animal since ancient times, and is popular. With the expansion of the culture scale, the trade of the soft-shelled turtle commodity and the seedling is frequent. Since 2007, a sudden death frequently occurs in turtle ecofarms. The diseased soft-shelled turtles are mainly young soft-shelled turtles with the weight of 150-. The disease season is 6-10 months, wherein 6-9 months are peak periods, the cumulative mortality rate in a disease pond within 10 days can reach 90-100%, and the disease pond is called turtle systemic septicemia (STSS) because the main symptoms are systemic hemorrhage and septicemia. The conventional drug treatment is ineffective, and brings huge economic loss to the breeding production. STSS is caused by the spherical virus STSSV of turtle systemic septicemia.

The traditional disease prevention mode in aquaculture is to blindly use chemical drugs, antibiotics and the like, which not only has no obvious effect, but also easily causes toxic reaction, seriously damages the ecological environment, induces drug resistance, and has drug residue to influence the safety of aquaculture products. In order to obtain more green foods beneficial to the health of consumers and protect the breeding environment for the purpose of continuous utilization, the exploration of a novel and effective antibiotic-free (antibiotic-free) prevention and treatment technology is urgent.

The life cycle of a virus-infected cell requires adsorption, invasion, uncoating, synthesis of viral components, assembly and release of virions. Adsorption and invasion are key links for determining the success of infection, and specific adsorption proteins on the surface of viruses and specific proteins or glycoproteins on the surface of target cells are required to be specifically recognized and combined. The blocking agent has important significance for preventing and treating viral diseases by preventing adsorption and invasion. The infection and pathogenic mechanism of STSSV are not known. At home and abroad, no report about a blocking inhibitor of the turtle systemic sepsis spherical virus is published.

Disclosure of Invention

The problems to be solved by the invention are as follows: the invention provides a recombinant turtle apolipoprotein B, which is used as a virus blocker, has inhibitory activity on STSSV infection, and can be used for developing a medicament for preventing and treating STSSV infection.

The technical scheme of the invention is as follows:

in the research, the turtle apolipoprotein B can be combined with STSSV. The invention designs a primer according to the nucleotide sequence and the protein functional domain of the turtle apolipoprotein B gene, clones partial gene segments of the turtle apolipoprotein B by taking turtle cDNA as a template, constructs prokaryotic expression recombinant plasmids, converts engineering bacteria, and induces the expression to obtain the recombinant turtle apolipoprotein B; assays were performed in which recombinant apolipoprotein B blocks stssv infection of target cells.

The sequence of the cloned recombinant turtle apolipoprotein B gene fragment is shown in a sequence table SEQ ID NO.1, and the amino acid sequence of the expressed recombinant turtle apolipoprotein B functional peptide fragment is shown in a sequence table SEQ ID NO. 2.

The STSSV turtle apolipoprotein B can inhibit STSSV from infecting liver cells and blood cells of a fish.

The preparation method of the STSSV turtle apolipoprotein B comprises the following steps:

cloning of partial gene of apolipoprotein B of STSSV turtle

Using an RNAiso plus (purchased from Dalibao Biopsis)Engineering company Limited) extracts total RNA from fresh turtle liver, and the extraction steps are carried out according to the instruction provided by Dalibao bioengineering company Limited; reverse transcription is carried out by taking total RNA as a template to obtain turtle cDNA, and PrimeScript is used in the reverse transcription process ^TM II 1st Strand cDNA Synthesis Kit (purchased from Dalibao bioengineering Co., Ltd.) according to the instructions; designing a plurality of primer pairs, wherein the active recombinant turtle apolipoprotein is finally obtained by using the amplification products of the following primer pairs: f5-CCGGAATTCCGCACACTGAAACTTCCTAAG-3' (the underlined position is the BamHI cleavage site) and R5-CCGCTCGAGTAACATCTCAGGAATTGTGATTGT-3' (HindIII cleavage site is underlined); PCR amplification is carried out by taking cDNA as a template, a specific DNA amplification strip is recovered, the recovered product is connected with pMD18-T (purchased from Dalibao bioengineering Co., Ltd.), E.coli DH5 alpha competent cell (purchased from Dalibao bioengineering Co., Ltd.) is transformed, 100 mu g/mL (Amp) of ampicillin is coated on a plate, a positive clone identified by PCR is inoculated in 5mL of LB liquid culture medium (1L of the culture medium contains 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, pH7.5), shaking overnight culture is carried out at 37 ℃ and 200rpm, and recombinant plasmid is extracted. The recombinant plasmid is sequenced to obtain a sequence shown in a table SEQ ID NO. 1.

2. Construction of recombinant expression vector for Escherichia coli

The recombinant T vector is subjected to double enzyme digestion by QuickCut BamHI and QuickCut HindIII (purchased from Dalibao bioengineering Co., Ltd.), electrophoresis, gel cutting and recovery of a turtle apolipoprotein B gene fragment, and the turtle apolipoprotein B gene fragment and an expression vector pET-28a are connected at 16 ℃ overnight by using T4 ligase (purchased from Dalibao bioengineering Co., Ltd.), so as to obtain the recombinant vector. E.coli DH 5. alpha. competent cells were transformed with the recombinant vector, spread on LB solid medium containing 50. mu.g/mL kanamycin (1 liter medium containing 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar, pH7.5), positive clones were selected and inoculated into 5mL LB medium, cultured overnight at 37 ℃ and 200rpm, and recombinant expression plasmids were extracted for use.

3. The preparation process of the construction Rosetta competent cell of the genetically recombinant Amyda sinensis apolipoprotein B Escherichia coli Rosetta genetically engineered bacterium is as follows:

(1) activation of Rosetta E.coli: taking a tube of laboratory preservation bacteria, thawing the laboratory preservation bacteria on ice, and inoculating the laboratory preservation bacteria on an LB solid culture medium in a streaking mode;

(2) the Rosetta E.coli single colony was picked from the LB plate and inoculated into 100mL LB medium containing 43. mu.g/mL chloramphenicol, followed by shaking culture at 37 ℃ and 200rpm until OD ₆₀₀ Stopping culturing when the value is about 0.35, and cooling the bacterial liquid in ice;

(3) centrifuging at 5000rpm for 10min to collect thallus;

(4) the pellet was treated with 40mL of pre-cooled 0.1M MgSO ₄ Suspending the solution, mixing uniformly, and standing for 10 min;

(5) centrifuging at 5000rpm for 10min to collect thallus;

(6) 40mL of precooled 0.1M CaCl in the pellet ₂ Suspending the solution, mixing uniformly, and standing for 10 min;

(7) centrifuging at 5000rpm for 10min to collect thallus;

(8) 1M CaCl containing 15% glycerol for cell precipitation ₂ The solution was gently suspended, dispensed in 100. mu.L/tube format, frozen at-80 ℃ in a freezer for use, and removed and frozen on ice.

And (3) transforming the recombinant expression plasmid prepared in the step (2) into escherichia coli Rosetta competent cells, coating the escherichia coli Rosetta competent cells on an LB (Luria-Lutken) plate containing 34 mu g/mL chloramphenicol and 50 mu g/mL kanamycin, carrying out PCR (polymerase chain reaction) positive identification on bacterial colonies by using F and R primers, and carrying out sequencing confirmation on an insert fragment in the positive bacterial colonies to obtain the escherichia coli genetic engineering bacteria carrying the turtle apolipoprotein B gene fragment.

4. Preparation of gene recombinant turtle apolipoprotein B

Selecting the positive colony obtained in the step 3, inoculating the positive colony in LB liquid culture medium containing chloramphenicol and kanamycin, and culturing at 37 ℃ and 200rpm overnight; inoculating the overnight cultured bacterial liquid into fresh culture medium at a ratio of 1: 100, and performing amplification culture at 37 deg.C and 200rpm to OD ₆₀₀ Adding isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 1mmol/l for induction expression, and continuously culturing for 4 hours; centrifuging at 5000rpm for 10min to collect thallus, and collecting thallus with Phosphate Buffer Solution (PBS) (1L water solution containing 8g sodium chloride, 0.2g potassium chloride, and dodecahydrate0.29g of disodium hydrogen phosphate, 0.2g of potassium dihydrogen phosphate and pH7.6), then centrifuging at 5000rpm for 10min, washing the thalli by centrifugation, and repeatedly washing the thalli once; adding 5mL of PBS (phosphate buffer solution) into the finally obtained thalli according to the wet weight per gram for resuspension, and ultrasonically crushing cells under the conditions that: 300W, break 2S, pause 8S, 120 cycles.

Centrifuging 12000g of the crushed bacterial liquid at normal temperature for 20min, collecting precipitates, resuspending the precipitates by using an inclusion body washing buffer solution (containing 20mmol/L Tris-HCl and 2mol/L urea), centrifuging 12000g of the precipitates for 20min, repeatedly washing the precipitates for multiple times, dissolving the finally obtained precipitates by using 5-10mL of urea buffer solution (containing 20mM Tris-HCl, 8M urea, 500mM NaCl and 5mM imidazole, adjusting the pH to 8.0, and adding 1mM beta-mercaptoethanol), and chromatographically purifying and eluting the dissolved inclusion body protein by using a His Cravitrap chelating column (purchased from GE company).

Putting the eluent into a dialysis bag, putting renaturation solution I (containing 0.1M Tris-HCl, reduced glutathione 0.614g, oxidized glutathione 0.0122g, 6M urea, glycerol 100mL and Tween-20100 mu L in 1L of aqueous solution) with the initial urea concentration of 6M into the dialysis bag for dialysis for 4 hours, and slowly introducing renaturation solution II (containing 0.1M Tris-HCl, reduced glutathione 0.614g, oxidized glutathione 0.0122g, glycerol 100mL and Tween-20100 mu L in 1L of aqueous solution) into the renaturation solution I in a siphoning manner by using a liquid delivery tube to carry out dialysis renaturation until the concentration of the external liquid urea is lower than 1M. Here, renaturation solution II was added at a flow rate of about 2mL per 5 minutes. And then, putting the dialysis bag into 0.1M Tris-HCl buffer solution for dialysis for 4 hours, then dialyzing in PBS for 2 hours, taking out the solution in the dialysis bag, centrifuging to remove the precipitate, and obtaining supernatant which is renatured recombinant turtle apolipoprotein B suspension. The molecular weight of the recombinant turtle apolipoprotein B is 48 kDa.

The detection of the effect of blocking STSSV infection of turtle cells by turtle apolipoprotein B comprises the following steps and results:

(1) fluorescein Isothiocyanate (FITC) labeled stssv: dissolving FITC powder in dimethyl sulfoxide (DMSO) solution to a final concentration of 1 mg/mL; incubating it with purified stssv for 1h at room temperature; centrifuging at 20000g at 4 deg.C for 20min, collecting precipitate, suspending the precipitate with PBS, centrifuging at 20000g at 4 deg.C for 20min again, and collecting precipitate; after four such washes, it was resuspended in 0.65% physiological saline.

(2) Making turtle liver cell prints and blood cell smears: taking a live turtle, smearing the turtle breastplate with alcohol cotton for disinfection, cutting soft tissues with a scalpel, cutting off artery blood vessels of the turtle with a dissecting scissors, dripping blood into a small beaker filled with an anticoagulant, gently mixing the blood vessels, centrifuging 500g, collecting cells, resuspending the cells with 0.65% physiological saline, taking a 10 mu L suspension smear, and fixing the smear by fire; dissecting the soft-shelled turtle, taking out the liver, slightly picking the surface film of the liver by using forceps, slightly printing the tissue blocks on a clean glass slide in a sheet printing mode, and fixing by fire.

(3) The recombinant turtle apolipoprotein B blocking test comprises the following steps: mixing FITC-labeled STSSV (FITC-STSSV) with a recombinant turtle apolipoprotein B solution, setting a mixed sample of FITC-STSSV and 0.65% physiological saline as a reference, and setting 0.65% physiological saline as a blank; and (3) respectively covering the liver cell printing sheet and the blood cell coating sheet prepared in the step (2), putting the liver cell printing sheet and the blood cell coating sheet on ice, incubating for 2 hours, repeatedly washing the glass sheet for 10 seconds by using 0.65% physiological saline, and sucking away residual water by using filter paper.

(4) 4', 6-diamidino-2-phenylindole (DAPI) staining: the slides were removed, DAPI was added to the cell area, maintained at room temperature for 3 minutes, rinsed with 0.65% saline, rinsed once with distilled water rapidly, and observed under a microscope for fluorescence. As a result, the FITC-STSSV signal of the cell added with the recombinant turtle apolipoprotein B is obviously weaker than that of the cell only added with FITC-STSSV, which indicates that the recombinant turtle apolipoprotein B can block the STSSV from infecting the turtle cell.

The invention has the beneficial effects that:

1. the invention provides a recombinant protein turtle apolipoprotein B, the molecular weight of which is 48 kDa.

2. The preparation process of the turtle apolipoprotein B comprises the steps of total RNA extraction, reverse transcription to obtain cDNA, primer design, PCR amplification, gene cloning, recombinant expression plasmid construction, Rosetta transformation, recombinant gene induced expression, recombinant protein purification and the like, can obtain the recombinant turtle apolipoprotein B with higher yield and purity, and has the characteristics of low cost, short period and easy operation.

3. The recombinant turtle apolipoprotein B can inhibit STSSV from infecting turtle cells.

4. The invention uses a prokaryotic expression system to prepare the recombinant turtle apolipoprotein B, inhibits STSSV from infecting turtle cells, and can be used as a blocking agent to develop a medicine for preventing and treating STSSV infection, thereby providing a new idea for the research and prevention and treatment of STSSV and having very important significance for the actual production.

Drawings

FIG. 1 shows electrophoresis of PCR products of apolipoprotein B gene fragment of Amyda sinensis. Lane M is a DL5000 DNA molecular weight standard, and

lanes

1 and 2 are DNA of apolipoprotein B gene fragment of Amyda sinensis obtained by PCR.

FIG. 2 shows SDS-PAGE electrophoresis of Rosetta engineering bacteria producing turtle apolipoprotein B. Lane M shows the protein molecular weight standard, lane 1 shows the Rosetta whole mycoprotein transferred with the recombinant plasmid but not induced, and lanes 2 to 6 show the Rosetta whole mycoprotein transferred with the recombinant plasmid and induced.

FIG. 3 shows SDS-PAGE electrophoresis of recombinant turtle apolipoprotein.

Lanes

1 and 2 are inclusion body proteins collected after sonicating Rosetta bacteria; lanes 3 to 5 are recombinant turtle apolipoproteins purified and renatured by nickel column.

FIG. 4 is a graph showing the effect of recombinant turtle apolipoprotein B in blocking FITC-STSSV from infecting turtle hepatocytes. FITC-excited green fluorescence signal represents STSSV virus, and DAPI-excited blue fluorescence represents nuclei. A. B, C line cells were treated with 0.65% physiological saline (blank), FITC-STSSV (control), FITC-STSSV + Trionyx sinensis Apolipoprotein B (experimental), respectively. Scale 10 μm.

FIG. 5 is a diagram showing the effect of recombinant turtle apolipoprotein B in blocking FITC-STSSV from infecting turtle blood cells. FITC-excited green fluorescence signal represents STSSV virus, and DAPI-excited blue fluorescence represents nuclei. A. B, C line cells were treated with 0.65% physiological saline (blank), FITC-STSSV (control), FITC-STSSV + Trionyx sinensis Apolipoprotein B (experimental), respectively. Scale bar 10 μm.

Detailed Description

Example 1

The preparation method of the recombinant turtle apolipoprotein B comprises the following specific steps:

1. recombinant turtle apolipoprotein B gene clone

1.1 extraction of Total RNA from Trionyx sinensis Wiegmann

Total RNA of the soft-shelled turtles was extracted by an RNA extraction kit (purchased from Dalibao bioengineering Co., Ltd.) according to the instructions.

1.2 reverse transcription to obtain turtle cDNA

PrimeScript was synthesized using the first Strand cDNA Synthesis kit ^TM II 1st Strand cDNA Synthesis Kit (purchased from Dalibao bioengineering Co., Ltd.) was reverse transcribed to obtain cDNA according to the instructions.

1.3 primer design

Designing a recombinant turtle apolipoprotein B gene cloning primer according to a whole gene sequence and a predicted functional domain of the turtle apolipoprotein B, introducing a restriction enzyme BamHI restriction site into an upstream primer F, and introducing a restriction enzyme HindIII restriction site into a downstream primer R. The primer was synthesized by Hangzhou Ongke Biotechnology Ltd. The primers are shown below:

F 5’-CCGGAATTCCGCACACTGAAACTTCCTAAG-3' (BamHI cleavage site in the dash)

R 5’-CCGCTCGAGTAACATCTCAGGAATTGTGATTGT-3' (HindIII restriction site in the underlined)

The PCR product obtained by amplification using the above primers is expected to be 1296bp in length.

Remarking: the primers are determined by many times of exploration, a plurality of pairs of primers are initially designed based on bioinformatics technology, and as a result, amplification products of other primers cannot express active protein in subsequent researches, and finally, the primers are successfully used.

1.4 cloning of recombinant Amyda sinensis apolipoprotein B Gene

PCR was performed using the cDNA in 1.2 as a template and the primers in 1.3. The reaction system is as follows:

the reaction conditions were as follows:

pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 30S, annealing at 56 ℃ for 35S, and extension at 72 ℃ for 1min for 30 cycles; extension at 72 ℃ for 10 min.

1.5 construction and identification of recombinant T vectors

Subjecting 5 μ L of the PCR product to 1 wt% agarose Gel electrophoresis, collecting the target fragment, and recovering the target fragment with a DNA purification Kit Gel Extraction Kit (purchased from Beijing Edelay Bio Inc.); connecting the recovered target fragment with a vector pMD18-T, transforming DH5 alpha competent cells, coating the competent cells on a plate containing 100 mu g/mL ampicillin (Amp), using the primer of 1.3 as a PCR to identify positive colonies, selecting the positive colonies, inoculating the positive colonies in 5mL LB liquid culture medium containing 100 mu g/mL Amp, and culturing at 37 ℃ and 200rpm overnight; extracting plasmid DNA by using a plasmid micro-extraction kit to obtain recombinant clone plasmid; taking the recombinant plasmid, determining the sequence of the insert fragment, and determining the correctness of the sequence.

The above connection conditions are: using pMD of Dalibao bioengineering Co., Ltd ^TM 18-T Vector Cloning Kit; the connection system comprises: 4.8 mu L of target DNA fragment, 0.2 mu L of pMD18-T vector and 5 mu L of solution I; ligation was carried out at 16 ℃ for 2 h.

The above conversion procedure is as follows:

(1) taking out the competent DH5 alpha frozen at-80 ℃, and placing on ice for thawing;

(2) adding 1 μ L of the above ligation product, gently rotating and mixing, and performing ice bath for 30 min;

(3) placing the ligation product and the mixture of DH5 alpha competent cells in a water bath at 42 ℃ for 90s by heat shock;

(4) quickly transferring to an ice bath, and cooling for 2 min;

(5) adding 800 μ L SOC culture medium, culturing at 37 deg.C and 200rpm for 1 h;

(6) 100 μ L of the above-mentioned bacterial suspension was taken out, spread on an LB solid medium plate containing 100 μ g/mL Amp, and subjected to inverted culture at 37 ℃ for 12 to 18 hours.

The SOC culture medium has the following formula:

1L of the medium contained 2% (W/V) tryptone, 0.5% (W/V) yeast extract, 0.05(W/V) NaCl, 2.5mM KCl, 10mM MgCl ₂ 20mM glucose。

The LB solid culture medium containing 100. mu.g/mL Amp has the following formula: the 1L culture medium contains 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar powder. Autoclaved, cooled to below 50 ℃ and Amp added to a final concentration of 100. mu.g/mL.

2. Construction of recombinant expression vector for Escherichia coli

2.1 recombinant clone T vector double digestion

The recombinant clone plasmid detected by sequencing is subjected to double enzyme digestion by using restriction enzymes QuiCutTM BamHI and QuiCutTM HindIII (purchased from Dalibao bioengineering Co., Ltd.), wherein the enzyme digestion conditions are as follows: water bath at 30 deg.c for 15min, and water bath at 37 deg.c for 15 min. The enzyme digestion system is as follows:

the enzyme digestion product is separated by 1 wt% agarose gel electrophoresis, and the target fragment is recovered by an agarose DNA recovery kit of Beijing Edley biology company, and the recovery method is according to the kit instruction.

2.2 expression vector double digestion

The expression vector pET-28a was double-digested with the restriction enzymes QuiCutTM BamHI and QuiCutTM HindIII. The enzyme digestion conditions are as follows: 30 ℃ in a water bath for 15min, and subsequently to 37 ℃ in a water bath for 15 min. The enzyme digestion system is as follows:

the digested products were separated by 1 wt% agarose gel electrophoresis, and the vector large fragment was recovered using agarose DNA recovery kit from Beijing Edley Biometrics.

2.3 construction and detection of expression vectors

Connecting the target fragment recovered after the enzyme digestion in the step 2.1 with the vector after the enzyme digestion in the step 2.2 to construct a recombinant expression vector, wherein the connection conditions are as follows: ligation was carried out overnight at 16 ℃. The linking system is as follows:

after ligation, the ligation products were taken to transform E.coli DH 5. alpha. competent cells, 100. mu.L of the transformed bacterial solution was spread on LB plate medium containing 50. mu.g/mL kanamycin (Kana), and the plate was placed in an incubator at 37 ℃ for inverted culture for 12-18 hours. Selecting a single colony, inoculating the single colony in an LB liquid culture medium containing 50 mu g/mL Kana, culturing overnight at the temperature of 37 ℃ and the rpm of 200, taking a positive bacterium solution identified by PCR, and extracting recombinant expression plasmids.

Remarking: the connection system and conditions in the process are determined by many times of exploration, the concentrations and the proportions of the enzyme digestion vector and the enzyme digestion target segment are explored through many times of experiments, and the connection temperature and time are finally successful.

Preparation of Rosetta competent cells

3.1 taking a tube of the laboratory preservative bacteria, thawing the tube on ice, and inoculating the tube to an LB solid culture medium in a streaking mode;

3.2 Single colonies of Rosetta E.coli were picked from the above LB plates, inoculated into 100mL of LB medium containing 43. mu.g/mL of chloramphenicol, and cultured at 37 ℃ with shaking at 200rpm until OD ₆₀₀ Stopping culturing when the value is about 0.35, and cooling the bacterial liquid in ice;

centrifuging at 3.35000 rpm for 10min, and collecting precipitate;

3.4 pellet of bacteria with 40mL of precooled 0.1M MgSO ₄ Suspending the solution, mixing uniformly, and standing for 10 min;

centrifuging at 3.55000 rpm for 10min to collect bacterial precipitate;

40mL of precooled 0.1M CaCl 3.6 pellet ₂ Suspending the solution, mixing uniformly, and standing for 10 min;

centrifuging at 3.75000 rpm for 10min to collect bacterial precipitate;

3.8 precipitation with 1M CaCl containing 15% Glycerol ₂ The solution was gently suspended, dispensed in 100. mu.L/tube format, frozen at-80 ℃ in a freezer for use, and removed and frozen on ice.

4. Construction of turtle apolipoprotein B escherichia coli genetic engineering bacteria

Transforming the recombinant expression plasmid prepared in the step 2 into escherichia coli Rosetta competent cells, coating the transformed product on an LB (Luria Bertani) plate containing 34 mug/mL of chloramphenicol and 50 mug/mL of Kana, placing the LB plate in an incubator at 37 ℃ for inverted culture for 12-18h, carrying out PCR (polymerase chain reaction) positive identification on bacterial colonies by using F and R primers, and carrying out sequencing confirmation on an insert fragment in the positive bacterial colonies to obtain the escherichia coli genetic engineering bacteria carrying the turtle apolipoprotein B gene fragment.

Wherein, the transformation step is similar to the step 1.5, and the main difference is that the plasmid is replaced by the recombinant expression plasmid prepared by 2.3, and the recipient bacterium is replaced by the competent Rosetta prepared by 3.

5. Induced expression of recombinant turtle apolipoprotein B

5.1 induced expression of Gene recombinant target protein Escherichia coli Gene engineering bacteria

The LB medium used in the following steps all contained 34. mu.g/mL chloramphenicol and 50. mu.g/mL Kana.

(1) Selecting the single colony, inoculating the single colony into 5mL LB culture medium, and culturing overnight at 37 ℃ and 200 rpm;

(2) 100. mu.L of the overnight-cultured broth was inoculated into 100mL of fresh LB medium and further cultured to OD ₆₀₀ The value of (A) is 0.6 to 0.7;

(3) adding isopropyl-beta-D thiogalactoside (IPTG) to a final concentration of 1mmol/L for induced expression for 4 h;

(4) 8000g of 1mL of bacterial liquid is taken to centrifugally collect thalli, and polyacrylamide gel electrophoresis (SDS-PAGE) analysis is carried out.

5.2 SDS-PAGE analysis of Amyda sinensis Apolipoprotein B expression

(1) Cleaning the glass plate, drying and installing;

(2) pouring 12% of separation gel: adding the components of the separation gel into a clean small beaker, uniformly mixing, immediately filling into a gap between glass plates, reserving a space required by the concentration gel (the tooth length of a comb is added with 1cm), and covering the liquid surface of the separation gel with 70% alcohol; the system of the separation gel is as follows:

(3) standing at room temperature for 30-60min, completely solidifying the separation gel, pouring out the covered distilled water, and sucking off the residual alcohol with filter paper;

(4) 5% concentrated gel perfusion: adding all components of the concentrated gel into a clean small beaker, uniformly mixing, immediately filling the mixture into the rest gaps of the glass plate, and slightly inserting a comb to avoid generating bubbles; the concentrated gel system is as follows:

(5) sample treatment: suspending 5.1 prepared thallus with 180 μ L of 1 × electrophoresis buffer (1L solution contains Tris 3.02g, glycine 14.4g, and SDS 1g), adding 60 μ L of 4 × protein loading buffer, boiling for 5min after vortex shaking, cooling, centrifuging for 10min at 120,000g, and taking supernatant;

(6) after the concentrated gel is completely solidified (about 30min), fixing the gel on an electrophoresis device, adding 1 × electrophoresis buffer solution inside and outside an electrophoresis tank, slightly pulling out a comb, and adding samples according to a preset sequence, wherein the sample loading amount of each hole is 20 μ L;

(7) connecting an electrophoresis device with a power supply, wherein the voltage applied to the gel is 80V, when the front edge of the dye enters the separation gel, the voltage is increased to 120V, the electrophoresis is continued until the dye reaches the bottom of the separation gel, and the power supply is turned off;

(8) taking out the gel, placing the gel in a small tray, adding Coomassie brilliant blue, placing the tray on a decoloring shaking table, carrying out shaking dyeing for 1.5h at room temperature, pouring out a dyeing solution, decoloring by using a decoloring solution (containing 150mL of methanol, 50mL of glacial acetic acid and sterile water for supplementing 500mL), and replacing the decoloring solution for several times until the strip is clear;

(9) the gel was removed, rinsed several times with distilled water, and then image analysis and photographic recording were performed with a gel imaging system. As a result, as shown in FIG. 2, the recombinant protein was expressed in Escherichia coli Rosetta with high efficiency.

6. Purification of recombinant turtle apolipoprotein B

Taking the bacteria liquid induced and expressed in 5.1, centrifuging at 8000g for 10min to collect thallus, resuspending the thallus with PBS with the same volume as the original bacteria liquid, centrifuging at 8000g for 10min to wash the thallus, and repeatedly washing the thallus 3 times. Adding 5mL of PBS (phosphate buffer solution) heavy suspension bacteria cells according to the wet weight of each gram of bacteria, performing ultrasonic bacteria crushing, wherein the crushing conditions are as follows: 300W, crush 2S, pause 8S, repeat 120 cycles. Centrifuging the crushed bacterial liquid for 20min at 12,000g, collecting a precipitate, suspending the precipitate by using an inclusion body washing buffer solution (containing 20mM Tris-HCl and 2M urea), centrifuging the crushed bacterial liquid for 20min at 12,000g, collecting the precipitate, suspending the precipitate by using an inclusion body washing buffer solution (containing 20mM Tris-HCl and 2M urea), centrifuging again, and repeatedly washing the precipitate for 5 times by using the inclusion body washing buffer solution in such a way to remove the foreign proteins as much as possible; the final centrifugation of the precipitate with 5-10mL 8M urea buffer solution, 4 ℃, 12,000g centrifugation for 15min collected supernatant, supernatant using His Gravitrrap affinity chromatography column (from GE company) according to the instructions for purification operation.

7. Renaturation of recombinant turtle apolipoprotein B

The purified protein solution of 6. was packed in a dialysis bag, and then sequentially placed in 500mL of PBS buffer solution containing 6M, 4M, 3M, 2M, 1.5M, 1M, and 0M urea or renaturation solution (1L of aqueous solution containing 50M Tris-HCl, 0.614g reduced glutathione, 0.0122g oxidized glutathione, 6M urea, 50mL glycerol, and 0. mu.L Tween-2050) for renaturation by dialysis, and the presence of the desired band was hardly detected by SDS-PAGE. Through multiple explorations, the formula of the renaturation solution is optimized, the rate of reducing the concentration of urea is reduced, and the following dialysis renaturation scheme is finally determined and is successful: and (6) filling the purified protein solution into a dialysis bag, putting the dialysis bag into 500mL of renaturation solution I (1L of aqueous solution contains 0.1M Tris-HCl, 0.614g of reduced glutathione, 0.0122g of oxidized glutathione, 6M urea, 0.5M glycine, 100mL of glycerol and 20100 mu L of Tween) for dialysis for 4h, and slowly introducing the renaturation solution II (1L of aqueous solution contains 0.1M Tris-HCl, 0.614g of reduced glutathione, 0.0122g of oxidized glutathione, 0.5M glycine, 100mL of glycerol and 20100 mu L of Tween) into the renaturation solution I in a siphoning manner by using an infusion tube until the concentration of urea in the external solution is lower than 0.5M. The flow rate of the renaturation solution II was about 2mL per 5 minutes. Then putting the dialysis bag into 0.1M Tris-HCl buffer solution for dialysis for 4h, then dialyzing in 0.1M PBS for 2h, taking out the solution in the dialysis bag, centrifuging to remove the precipitate, and collecting the supernatant containing renatured recombinant turtle apolipoprotein B (figure 3).

Example 2

The experiment for blocking virus infection of target cells by the recombinant turtle apolipoprotein B comprises the following steps:

(1) fluorescein Isothiocyanate (FITC) labeled stssv: FITC powder was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 1 mg/mL. Incubating the purified STSSV with a DMSO solution containing FITC for 1h at room temperature, centrifuging at 20,000g for 20min at 4 ℃, collecting the precipitate, suspending the precipitate with PBS, centrifuging at 20000g for 20min again at 4 ℃, and collecting the precipitate; after four times of such washing, it was resuspended in 0.65% physiological saline;

(2) preparing blood cell smear and turtle liver cell printing sheet: taking a live turtle, disinfecting a turtle breastplate with alcohol cotton, cutting soft tissues with a scalpel, cutting an artery vessel of the turtle with a dissecting scissors, dripping blood into a small beaker filled with an anticoagulant, gently mixing the blood and the blood, centrifuging 500g, collecting cells, resuspending the cells with 0.65% physiological saline, taking a 10 mu L suspension smear, and fixing the smear by fire; dissecting the soft-shelled turtle, taking out the liver, slightly opening the thin film on the surface of the liver by using forceps, slightly printing the thin film on a clean glass slide in a sheet printing mode, and fixing the thin film on the glass slide by fire;

(3) stssv incubation of turtle cells: mixing FITC-labeled STSSV (FITC-STSSV) with a recombinant turtle apolipoprotein B solution, setting FITC-STSSV and 0.65% physiological saline as a contrast, and setting 0.65% physiological saline as a blank; covering the liver cell printing sheet and the blood cell coating sheet prepared in the step (2) with the water solution respectively, putting the liver cell printing sheet and the blood cell coating sheet on ice for incubation for 2 hours, repeatedly washing the glass sheet for 10 seconds by using 0.65% physiological saline, and sucking away residual water by using filter paper;

(4) DAPI staining: the slide was removed, washed with 0.65% physiological saline, residual water was removed with filter paper, DAPI was added to the cell area, maintained at room temperature for 3 minutes, washed with 0.65% physiological saline, washed rapidly with distilled water, and observed under a microscope for fluorescence.

The results are shown in FIGS. 4 and 5. By comparing the fluorescence signals of the experimental group and the negative control group, the green fluorescence signals of the cells of the negative control group are stronger no matter the cells are blood cells or liver cells, but the fluorescence signals can hardly be detected by the cells of the experimental group, which shows that the viruses adsorbed by the cells of the experimental group are fewer, and shows that the recombinant protein turtle apolipoprotein B can block the combination of STSSV and the cells. The recombinant turtle apolipoprotein B is proved to have obvious inhibition effect on the infection activity of STSSV, and can be used for developing a medicament for preventing and treating STSSV.

Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that such changes, modifications, additions and substitutions are within the true spirit of the invention.

Claims

1. The recombinant turtle apolipoprotein B is characterized by comprising the following components: the protein is a turtle recombinant apolipoprotein B expressed by pronucleus, the nucleic acid sequence corresponding to the recombinant protein is the sequence listed in a sequence table SEQ ID NO.1, and the amino acid sequence is the sequence listed in a sequence table SEQ ID NO.2, so that the infection of the turtle systemic septicemia spherical virus can be blocked.

2. The method of claim 1 for the preparation of recombinant apolipoprotein B from turtles, comprising the steps of:

(1) recombinant turtle apolipoprotein B gene clone

Extraction of total RNA from soft-shelled turtle

Extracting total RNA of the soft-shelled turtle by adopting an RNA extraction kit RNAiso plus according to an instruction;

② reverse transcription to obtain turtle cDNA

Performing reverse transcription by adopting a first chain cDNA Synthesis Kit PrimeScript II 1st Strand cDNA Synthesis Kit according to the instruction;

design of primer

Designing a recombinant turtle apolipoprotein B gene cloning primer according to a whole gene sequence and a predicted functional domain of the turtle apolipoprotein B, introducing a restriction enzyme BamHI restriction site into an upstream primer F, and introducing a restriction enzyme HindIII restriction site into a downstream primer R; the primer sequences are shown as follows:

F 5’-CCGGAATTCCGCACACTGAAACTTCCTAAG-3' is underlined a BamHI site

R 5’-CCGCTCGAGTAACATCTCAGGAATTGTGATTGT-3' is underlined a HindIII site

Cloning of recombinant turtle apolipoprotein B gene

Using cDNA in step two as a template, and using primers in step three to perform PCR amplification, wherein the reaction system is as follows:

the reaction conditions were as follows:

pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 30S, annealing at 56 ℃ for 35S, and extension at 72 ℃ for 1min for 30 cycles; extending for 10min at 72 ℃;

fifthly, constructing and identifying the recombinant T vector

Carrying out 1 wt% agarose Gel electrophoresis on the PCR product, cutting a target fragment, and recovering the target fragment by using a DNA purification Kit Gel Extraction Kit; connecting the recovered target fragment with a vector pMD18-T, transforming DH5 alpha competent cells, coating the competent cells on a plate containing 100 mu g/mL ampicillin Amp, using the primer of 'primer design' to perform PCR, identifying positive colonies, selecting the positive colonies, inoculating the positive colonies in 5mL LB liquid culture medium containing 100 mu g/mL Amp, and culturing at 37 ℃ and 200rpm overnight; extracting plasmid DNA by using a plasmid micro-extraction kit to obtain recombinant clone plasmids; taking the recombinant plasmid, determining the sequence of the insert fragment, and determining the correctness of the sequence;

the above connection conditions are: using pMD18-T Vector Cloning Kit; the connecting system comprises the following components: 4.8 mu L of target DNA fragment, 0.2 mu L of pMD18-T vector and 5 mu L of solution I; connecting for 2h at 16 ℃;

the above transformation procedure is as follows:

A. taking out the competent DH5 alpha frozen at-80 ℃, and placing on ice for thawing;

B. adding 1 μ L of the above ligation product, gently rotating and mixing, and performing ice bath for 30 min;

C. placing the ligation product and the mixture of DH5 alpha competent cells in a water bath at 42 ℃ for 90s by heat shock;

D. quickly transferring to an ice bath, and cooling for 2 min;

E. adding 800 μ L SOC culture medium, culturing at 37 deg.C and 200rpm for 1 h;

F. taking out 100 μ L of the above bacterial liquid, spreading on LB solid culture medium plate containing 100 μ g/mL Amp, and performing inverted culture at 37 deg.C for 12-18 h;

(2) construction of recombinant expression vector for Escherichia coli

Recombinant clone T vector double enzyme digestion

Carrying out double enzyme digestion on the recombinant clone plasmid subjected to sequencing detection by using restriction enzymes QuiCut BamHI and QuiCutHindIII, wherein the enzyme digestion conditions are as follows: water bath at 30 deg.C for 15min, and transferring to water bath at 37 deg.C for 15 min; the enzyme digestion system is as follows:

carrying out electrophoresis separation on the enzyme digestion product by 1 wt% agarose gel, and recovering the target fragment by using an agarose DNA recovery kit, wherein the recovery method is according to the kit specification;

② double digestion of expression vector

Carrying out double enzyme digestion on the expression vector pET-28a by using restriction enzymes QuiCut BamHI and QuiCut HindIII; the enzyme digestion conditions are as follows: water bath at 30 deg.C for 15min, and then transferring to water bath at 37 deg.C for 15 min; the enzyme digestion system is as follows:

carrying out electrophoresis separation on the enzyme digestion product by 1 wt% agarose gel, and recovering a large carrier fragment by using an agarose DNA recovery kit;

construction and detection of expression vector

Connecting the target fragment recovered after enzyme digestion in the first step with the vector after enzyme digestion in the second step to construct a recombinant expression vector, wherein the connection conditions are as follows: ligation was carried out overnight at 16 ℃ in the following manner:

after connection, taking a connection product to transform escherichia coli DH5 alpha competent cells, taking 100 mu L of bacterial liquid after transformation to coat on an LB plate culture medium containing 50 mu g/mL kanamycin Kana, placing the plate in a 37 ℃ culture box for inverted culture for 12-18h, selecting a single bacterial colony to inoculate in an LB liquid culture medium containing 50 mu g/mL Kana at 37 ℃, culturing overnight at 200rpm, taking positive bacterial liquid identified by PCR, and extracting recombinant expression plasmids;

(3) preparation of Rosetta competent cells

Firstly, taking a tube of laboratory preservative bacteria, thawing the tube on ice, and inoculating the tube onto an LB solid culture medium in a streaking mode;

② single colony of Rosetta Escherichia coli is selected from the LB plate and inoculated into 100mL LB culture medium containing 43 mug/mL chloramphenicol, 37 ℃, 200rpm vibration culture to OD ₆₀₀ Stopping culturing when the value is 0.35, and cooling the bacterial liquid in ice;

thirdly, centrifuging at 5000rpm for 10min and collecting precipitates;

fourthly, the bacterial sediment is pre-cooled by 40mL of 0.1M MgSO ₄ Suspending the solution, mixing uniformly and standing for 10 min;

centrifuging at 5000rpm for 10min to collect bacteria precipitate;

sixthly, 40mL precooled 0.1M CaCl is precipitated ₂ Suspending the solution, mixing uniformly, and standing for 10 min;

seventhly, centrifuging at 5000rpm for 10min to collect bacterial precipitates;

allowing 1M CaCl containing 15% glycerol to precipitate ₂ Slightly suspending the solution, subpackaging with 100 μ L/tube, freezing in-80 deg.C refrigerator for use, taking out, and thawing on ice;

(4) construction of turtle apolipoprotein B escherichia coli genetic engineering bacteria

Transforming the recombinant expression plasmid prepared in the step (2) into escherichia coli Rosetta competent cells, coating the transformed product on an LB (Luria Bertani) plate containing 34 mug/mL of chloramphenicol and 50 mug/mL of Kana, putting the LB plate in a 37 ℃ incubator for inverted culture for 12-18h, growing a single colony which is escherichia coli genetic engineering bacteria, and determining and confirming the inserted turtle apolipoprotein gene sequence;

wherein, the operation of transformation is similar to the transformation of the fifth step in the step (1), the main difference is that the plasmid is replaced by the recombinant expression plasmid prepared in the step (2), and the recipient bacterium is replaced by Rosetta;

(5) induced expression of recombinant turtle apolipoprotein B

The LB medium used in the following steps all contained 34. mu.g/mL chloramphenicol and 50. mu.g/mL Kana

A. Selecting the single bacterial colony of the engineering bacteria prepared in the step (4) to inoculate into 5mL LB culture medium, culturing overnight at 37 ℃ and 200 rpm;

B. 100. mu.L of the overnight-cultured bacterial suspension was inoculated into 100mL of fresh LB medium and cultured until OD was reached ₆₀₀ The value of (A) is 0.6 to 0.7;

C. adding isopropyl-beta-D thiogalactoside IPTG to a final concentration of 1mmol/L for induced expression for 4 h;

D. collecting 8000g of bacteria liquid in 1mL in a centrifugal mode, and carrying out polyacrylamide gel electrophoresis (SDS-PAGE) analysis on the collected bacteria;

(6) purification of recombinant turtle apolipoprotein B

Taking the induced bacteria liquid prepared in the step (5), centrifuging at 8000g for 10min to collect thalli, resuspending the thalli by PBS with the same volume as the original bacteria liquid, centrifuging at 8000g for 10min to wash the thalli, repeatedly washing the thalli for 3 times, adding 5mL PBS resuspension bacteria cells according to the wet weight of each gram of the thalli, and performing ultrasonic bacteria crushing under the conditions of: 300W, crushing for 2S, pausing for 8S, and repeating 120 cycles; centrifuging the crushed bacterial liquid for 20min at 12,000g, collecting a precipitate, suspending the precipitate by using an inclusion body washing buffer solution, centrifuging for 20min at 12,000g, collecting the precipitate, suspending the precipitate by using the inclusion body washing buffer solution, centrifuging again, and repeatedly washing for 5 times by using the inclusion body washing buffer solution in the way so as to remove the impure protein as far as possible, wherein the formula of the inclusion body washing buffer solution is as follows: containing 20mM Tris-HCl and 2M urea; centrifuging the precipitate obtained by the last centrifugation with 5-10mL of 8M urea buffer solution at 4 ℃ for 15min at 12,000g, collecting the supernatant, and purifying the supernatant with His Gravitrrap affinity chromatography column according to the instruction;

(7) renaturation of recombinant turtle apolipoprotein B

Putting the protein solution purified in the step (6) into a dialysis bag, putting the dialysis bag into 500mL of renaturation solution I for dialysis for 4 hours, and slowly introducing the renaturation solution II into the renaturation solution I by using a liquid conveying pipe in a siphoning mode at the flow rate of 2mL per 5 minutes until the concentration of external liquid urea is lower than 0.5M; then putting the dialysis bag into 0.1M Tris-HCl buffer solution for dialysis for 4h, then dialyzing in 0.1M PBS for 2h, taking out the solution in the dialysis bag, centrifuging to remove the precipitate, and obtaining supernatant containing renatured recombinant turtle apolipoprotein B, namely the target protein in claim 1; the formula of the renaturation liquid I is as follows: 1L of water solution contains 0.1M Tris-HCl, 0.614g of reduced glutathione, 0.0122g of oxidized glutathione, 6M urea, 0.5M glycine, 100mL of glycerol and 20100 mu L of Tween; the formula of the renaturation solution II is that 1L of aqueous solution contains 0.1M Tris-HCl, 0.614g of reduced glutathione, 0.0122g of oxidized glutathione, 0.5M glycine, 100mL of glycerol and 20100 mu L of Tween.

3. The application of the turtle apolipoprotein B of claim 1 in preparing a virus blocking agent for blocking STSSV infection, wherein the virus blocking agent is an antiviral drug and a feed additive.