CN113234761A - Preparation method of baculovirus capable of soluble expressing toxoplasma MIC1 recombinant protein - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to a preparation method of a baculovirus of a soluble expression toxoplasma MIC1 recombinant protein, which comprises the steps of designing a specific primer through a toxoplasma cDNA sequence, amplifying a target fragment, connecting the target fragment to a pET-22b vector after double enzyme digestion, amplifying the primer, connecting an insect cell expression vector pFastBac1 through double enzyme digestion, and preparing a connection product pFastBac1-MIC 1. Transforming to DH10Bac competent cells, screening and separating by resistant blue-white spots to obtain a recombinant baculovirus shuttle vector rebamid-MIC 1, transfecting Sf9 insect cells, and subculturing to obtain the recombinant baculovirus. The recombinant baculovirus can express soluble MIC1 recombinant protein in the replication process, and can be used for the subsequent development of antibodies and biological functions thereof.

Description

Preparation method of baculovirus capable of soluble expressing toxoplasma MIC1 recombinant protein

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a preparation method of a baculovirus capable of soluble expressing Toxoplasma gondii MIC1 recombinant protein.

Background

Toxoplasma gondii is an obligate parasitic parasite within the nucleated cells, without host specificity, and can infect humans and almost all warm-blooded animals. Toxoplasma infections are caused by ingestion of food and water contaminated with the parasite resistant stage (oocysts), or by consumption of uncooked meat containing its encapsulated stage. Primary infections in humans are usually asymptomatic, or cause mild symptoms in a host with an intact immune system. However, toxoplasmosis in immunosuppressed patients can cause severe damage due to reactivation of latent cysts. The toxoplasmosis has brought great harm to human and livestock breeding because of no specific treatment medicine.

In the process of invading host cells by toxoplasma, the microlin plays an extremely important role, the microlin is a secretory organ existing at the front end of the toxoplasma, reaches the microlin through the Golgi body after being synthesized in a rough endoplasmic reticulum, and is secreted out of the body, and more than 15 kinds of currently known microlins comprise MIC1, MIC12, AMA1 and the like. During secretion, transport and release of the microgrins, they all bind to additional microgrins and act in the form of complexes. Toxoplasma gondii microgrinin 1(TgMIC1) was obtained by monoclonal antibody screening. MIC1 contains no transmembrane region, the protein sequence contains 18 cysteine residues, and cysteine regions are frequently found in receptor molecules, suggesting that MIC1 may play a role in the binding of toxoplasma to the host cell surface.

Common protein expression systems can be divided into prokaryotic expression systems and eukaryotic expression systems, and an escherichia coli expression system is the most developed prokaryotic expression system at present, has the advantages of good operability of prokaryotic cells, clear genetic background, low cost, relatively simple separation and purification of expression products and the like, but cannot be subjected to glycosylation modification, lacks a processing mechanism after protein translation, such as formation of disulfide bonds and the like, and is easy to generate endotoxin. The insect baculovirus expression system is one kind of eukaryotic expression system with unique biological characteristic, and has post-translational modification and foreign protein transferring capacity similar to that of most higher eukaryotic organisms, so that it is suitable for most of proteins. The insect baculovirus expression system is a very popular eukaryotic expression system at home and abroad at present, and an expression vector constructed by using a strong promoter of polyhedrin in a baculovirus structural gene can enable a plurality of eukaryotic target genes to be expressed effectively and even at a high level. Because the separation of the recombinant virus is through blue white spot screening, the problem of wild type and non-recombinant virus pollution does not exist, and the recombinant virus has the advantages of higher eukaryotic expression systems, such as the formation of disulfide bonds and the like which are functions of post-translational processing modification, so that the recombinant protein is closer to natural protein in structure and function. The maximum expression amount can reach 50% of the total amount of insect cell protein. Very large exogenous genes can be expressed. Baculovirus can accommodate large molecular inserts and can package large gene fragments. The baculovirus expression system has the capacity of expressing a plurality of genes simultaneously in the same cell, and can adopt different recombinant viruses to infect the cell simultaneously and clone two exogenous genes simultaneously on the same transfer vector. The expression product can be processed to form active heterodimers or multimers. Insect cells can reach higher cell density in culture, which is beneficial to large-scale expression of recombinant proteins and is an ideal way for expressing bioactive proteins.

Disclosure of Invention

Therefore, the invention provides a preparation method of baculovirus for soluble expression of Toxoplasma gondii MIC1 recombinant protein, so as to achieve the effect of high-efficiency soluble expression of Toxoplasma gondii MIC1 in vitro.

In order to achieve the above purpose, the invention provides the following technical scheme:

the preparation method of the baculovirus for soluble expression of Toxoplasma gondii MIC1 recombinant protein comprises the following steps:

s1, cutting 1-72 bit sequence of the gene sequence coding signal peptide of toxoplasma gondii microglial TGME49_291890 to be used as a target gene, and obtaining a sequence shown as SEQ1 to be used as the target gene;

s2, designing primers MIC1-NdeI-5 and MIC1-XhoI-3 with enzyme cutting sites for MIC 1;

s3, using cDNA of a target gene as a template, amplifying a target gene fragment by using the primer in the step S2, carrying out double enzyme digestion by NdeI and XhoI to obtain a target fragment with an enzyme digestion site, carrying out double enzyme digestion on the pET22b vector by using the NdeI and the XhoI, respectively purifying and recovering the target fragment after enzyme digestion and a linearized vector, and connecting the target fragment and the linearized vector through ligase overnight to obtain a connection product pET22b-MIC 1;

s4, designing primers MIC1-XbaI-5 and MIC1-Hind III-5 with enzyme cutting sites for pET22b-MIC 1;

s5, using pET22b-MIC1 as a template, adopting the primer in the step S4 to amplify a target fragment, performing double enzyme digestion by Hind III and XbaI to obtain the target fragment with an enzyme digestion site, performing double enzyme digestion by Hind III and XbaI to obtain an insect cell expression vector pFastBac1, respectively purifying and recovering the target fragment after enzyme digestion and a linearized vector, and performing a connection reaction on the target fragment and the linearized vector through ligase to obtain a connection product pFastBac1-MIC1 transfer vector;

s6, transforming the connecting product pFastBac1-MIC1 transfer vector to a DH10Bac competent cell, and obtaining a recombinant baculovirus shuttle vector rebamid-MIC 1 through resistant blue-white spot screening and separation;

s7, transfecting the recombinant baculovirus shuttle vector rebamid-MIC 1 to Sf9 insect cells, and carrying out three-time cell subculture to obtain the recombinant baculovirus.

Further, in step S2, the primer sequence is:

MIC1-NdeI-5：5'-CCTTCATATGGCGTCGCATTCTCAT-3', the restriction sites NdeI are underlined;

MIC1-XhoI-3：5'-TAACCTCGAGAGCAGAGACGGCCG-3', the cleavage site XhoI is underlined.

Further, the amplification in step S3 is PCR amplification; the PCR amplification reaction parameters are as follows: pre-denaturation at 98 ℃ for 1min, denaturation at 98 ℃ for 10s, annealing at 47 ℃ for 15s, and extension at 72 ℃ for 10s, after 10 cycles; denaturation at 98 ℃ for 10s, annealing at 69 ℃ for 15s, extension at 72 ℃ for 10s, re-extension at 4 ℃ and 25 cycles.

Further, the temperature of the overnight ligation in step S3 was 16 ℃.

Further, the temperature of the enzyme digestion in the step S3 is 37 ℃ and the time is 30 min.

Further, in step S3, the amplification process includes buffer solution of the reaction product, NdeI, XhoI, template, dd H₂The volume ratio of O is 25:2:2:2: 19; the ligase in the ligation reaction process is T4 DNA, wherein the volume ratio of reactant T4 DNA ligase, 10 × Buffer, vector and target gene is 1:1:1: 7.

Further, in step S4, the primer sequence is:

MIC1-XbaI-5：5'-TTAATCTAGAATGGCGTCGCATTCTC-3', the restriction site XbaI is underlined;

MIC1-HindⅢ-5：5'-TTGCAAGCTTTCAGTGGTGGTGG-3', the restriction site Hind III is underlined.

Further, the amplification in step S5 is PCR amplification; PCR amplification reaction parameters: pre-denaturation at 98 ℃ for 1min, denaturation at 98 ℃ for 15s, annealing at 47 ℃ for 15s, extension at 72 ℃ for 20s, re-extension at 4 ℃ and 35 cycles.

Further, in the amplification process described in step S5, the reaction buffer, NdeI, XhoI, template, dd H₂The volume ratio of O is 25:2:2:2: 19; the ligase in the ligation reaction process is T4 DNA, wherein the volume ratio of reactant T4 DNA ligase, 10 × Buffer, vector and target gene is 1:1:1: 7.

Further, the MIC1 recombinant protein expressed by the recombinant baculovirus in insect cells has solubility and antigenicity.

Compared with the prior art, the invention has the beneficial effects that:

the recombinant baculovirus of the invention can successfully express recombinant protein in insect cells by utilizing the presentation effect of the baculovirus, and the MIC1 recombinant protein in the insect cells keeps the antigenicity and solubility due to accurate modification and correct folding after translation, so that the recombinant baculovirus can be used for the subsequent development of antibodies and biological functions thereof.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 shows the result of amplification of an MIC1 target fragment provided by the present invention;

mark is Trans 2K PLUS DNA Marker, and the size of the target fragment is 1300 bp;

wherein M is a DNA molecular weight standard; 1, 2, 3 and 4 are MIC1 amplification products.

FIG. 2 shows the result of double restriction enzyme identification of pET22b-MIC1 plasmid provided by the present invention;

mark is Trans 2K PLUS DNA Marker, pET22b vector is 5493bp, and the size of target fragment is 1300 bp;

wherein M is a DNA molecular weight standard; 1 is the single enzyme cutting identification result, and 2 is the double enzyme cutting identification result of pET22b-MIC1 plasmid.

FIG. 3 shows the result of the double restriction enzyme digestion of plasmid pFast bac1-MIC 1;

mark is Trans 2K PLUS DNA Marker, pFastBac1 vector is 4755bp, and the size of the target fragment is 1300 bp;

wherein M is a DNA molecular weight standard; 1, 2, 3 and 4 are double enzyme digestion identification results of pFastBac1-MIC1 vectors.

FIG. 4 shows the result of PCR identification of recombinant baculovirus-containing plasmids screened by blue-white screening provided by the present invention;

wherein, M is DNA molecular weight standard, 1, 2, 3 and 4 are recombinant baculovirus plasmid DNA amplification products extracted from white colonies, and 5 is recombinant baculovirus plasmid DNA amplification products extracted from blue colonies.

FIG. 5 shows PCR verification of baculovirus genomic DNA extracted from the supernatant of the inoculated/normal cells provided by the present invention; in order to ensure the accuracy of the experiment, the experiment is repeatedly carried out twice, and the result is verified by PCR;

wherein, M is DNA molecular weight standard, 1 is supernatant of inoculated cells, and 2 is supernatant of blank control cells.

FIG. 6 shows the result of the bright field test for the expression of IFA by the MIC1 of the virus-inoculated cell provided by the invention.

FIG. 7 shows the result of DAPI staining for the expression of IFA by the MIC1 of the virus-inoculated cell provided by the invention.

FIG. 8 shows the result of detecting mouse anti-MIC 1 monoclonal antibody by using the MIC1 expression IFA of the virus-inoculated cell provided by the invention.

FIG. 9 shows the results of the detection of expression of IFA in MIC1 of normal cells in bright field.

FIG. 10 shows the result of DAPI staining for the expression of IFA in MIC1 of normal cells provided by the present invention.

FIG. 11 shows the result of detecting mouse anti-MIC 1 monoclonal antibody by normal cell MIC1 expression IFA provided by the invention.

FIG. 12 shows the Western Blot identification of MIC1 expression of the virus-infected cells provided by the invention;

wherein, M is protein Mark, 1 is normal Sf9 cell lysate, and 2 is virus-inoculated Sf9 cell lysate.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Experimental Material

DH10Bac competent cells for preventive veterinary medicine research laboratory preservation of animal husbandry and veterinary medicine college of Shenyang agriculture university

The prokaryotic expression vector pET22b and the insect cell expression vector pFastBac1 are preserved in the preventive veterinary medicine teaching and research room of the livestock and veterinary medicine school of Shenyang agriculture university

Sf9 insect cells for preventive veterinary medicine textbook room preservation of Shenyang agriculture university stockbreeding veterinary school

The endonucleases NdeI, XhoI, HindIII and XbaI TaKaRa

T4 DNA ligase TaKaRa Co

Small plasmid extraction kit TaKaRa Co

Tiangen Co of blood/cell/tissue genome DNA extraction kit

SF900 II serum-free Medium Gibco, USA

LipoInsect^TMBiyuntian Co Ltd, transfection reagent

Bacmid mini extraction kit Biyuntian Co of baculovirus shuttle vector bacmid

Biyuntian Co Ltd with 5 xSDS-PAGE sample buffer

HRP-labeled goat anti-mouse IgG (H + L) Biyuntian Co

Ku Laibobo technology Co Ltd in Beijing of Glycine

Bovine serum Albumin V (BSA) Solebao Co

Alexa flow @488 coat anti-rabbitig (H + L) Shanghai Invitrogen corporation

ProLong Gold antibody mountain with DAPI Thermon Fisher corporation

CD71 Cell Signaling, murine monoclonal antibody

DNA Standard Mark Beijing Quanji Co

Trans1-T1 competent cell, Beijing Quanjin Co

Example 1 construction of recombinant baculovirus containing MIC1 Gene

Step one, after logging in a Toxo DB website and searching TGME49_291890, analyzing a gene sequence and a protein sequence, downloading a full-length gene sequence, and cutting a sequence (1-72 bits) for encoding a signal peptide: primers with enzyme cutting sites for MIC1 are designed as shown in SEQ1, and the primers are as follows:

MIC1-NdeI-5：5'-CCTTCATATGGCGTCGCATTCTCAT-3'；

MIC1-XhoI-3：5'-TAACCTCGAGAGCAGAGACGGCCG-3'；

synthesis of cDNA: s1, extraction of Toxoplasma gondii RH strain total RNA

Extracting total RNA of toxoplasma through Trizol reagent, which comprises the following steps:

1) removing the purified insect body precipitate (about 1 × 10) from the ultra-low temperature refrigerator at-80 deg.C⁸) Sucking 1mL of Trizol reagent, re-suspending the insect body precipitate, and repeatedly blowing the precipitate uniformly to ensure that the insect body precipitate is completely cracked;

2) adding 200 mu L of chloroform into the Trizol mixed solution, violently shaking for 15-20 s in a vortex oscillator, and standing for 5min at room temperature; (after standing, the mixed solution is obviously layered, the upper layer is colorless clear solution, and the lower layer is red);

3) placing the mixed solution in a 4 ℃ low-temperature centrifuge, centrifuging for 15min at 12000 Xg, sucking the supernatant into a new 1.5mL centrifuge tube after the centrifugation is finished, wherein the sucked supernatant is about 500 mu L;

4) adding isopropanol solution with the same volume into the supernatant, fully and uniformly mixing, reversing for 1-2 min, and standing at-20 ℃ for more than 30 min;

5) at this time, a small amount of precipitate appeared in the 1.5mL centrifuge tube, and the precipitate was nucleic acid fragments; placing the centrifuge tube in a 4 deg.C low temperature centrifuge, centrifuging at 12000 Xg for 20min, and discarding the supernatant to obtain precipitate;

6) adding 1mL of 75% ethanol solution into the precipitate, violently shaking in a vortex oscillator for 30s, placing in a 4 ℃ low-temperature centrifuge, centrifuging at 7500 Xg for 5min, and removing the supernatant;

7) adding 1mL of 75% ethanol solution, violently shaking in a vortex oscillator for 30s, placing in a 4 ℃ low-temperature centrifuge, centrifuging at 12000 Xg for 5min, and removing the supernatant;

8) opening a centrifugal tube, and standing at room temperature for 3-5 min to completely volatilize the residual ethanol; and then adding 20-30 mu L DEPC deionized water to completely dissolve the precipitate, then placing the precipitate in ice to measure the concentration, and entering a reverse transcription experimental process.

S2, cDNA synthesis: the experimental procedure was carried out with reference to a reverse transcription kit from Monscript RTIII All-in-One Mix with dsDNase REF: MR 05101. The cDNA was stored at-20 ℃.

Amplifying a target gene fragment, the target fragment subjected to double enzyme digestion amplification and a pET22b vector by taking cDNA as a template, respectively purifying and recovering the target fragment subjected to enzyme digestion and the linearized vector, and connecting overnight at 16 ℃ to obtain pET22b-MIC 1; wherein, the amplification reaction parameters are shown in Table 1, the enzyme digestion reaction parameters are shown in Table 2, and the ligation reaction parameters are shown in Table 3.

TABLE 1 PCR amplification reaction System

First stage	Pre-denaturation	Denaturation of the material	Annealing	Extension
						10 cycles	98℃	98℃	47℃	72℃
	1min	10s	15s	10s
						Second stage		Denaturation of the material	Annealing	Extension	Re-extension of
25 cycles		98℃	69℃	72℃	4℃
								10s	15s	10s	-∞

TABLE 2 restriction system

Total volume	50μL
		Buffer	25μL
NdeI	2μL
		XhoI	2μL
Form panel	2μL
		dd H2O	19μL
Conditions of enzyme digestion	37℃，30min

TABLE 3 ligation reaction System

Step two, designing a primer with an enzyme cutting site for pET22b-MIC1, wherein the primer is as follows:

MIC1-XbaI-5：5'-TTAATCTAGAATGGCGTCGCATTCTC-3'

MIC1-HindⅢ-5：5'-TTGCAAGCTTTCAGTGGTGGTGG-3'

amplifying a target fragment by taking pET22b-MIC1 as a template, obtaining the target fragment with a restriction enzyme cutting site by HindIII and XbaI double restriction enzyme cutting, utilizing HindIII and XbaI double restriction enzyme cutting insect cell expression vector pFastBac1, respectively purifying, recovering and cutting the target fragment after restriction enzyme cutting and a linearization vector, and carrying out connection reaction on the target fragment and the linearization vector by T4 DNA ligase to obtain a connection product pFastBac1-MIC1 transfer vector; the amplification reaction parameters are shown in Table 4, the digestion reaction parameters are shown in Table 5, and the ligation reaction parameters are shown in Table 6.

TABLE 4 PCR reaction System

35 cycles	Pre-denaturation	Denaturation of the material	Annealing	Extension	Re-extension of
							98℃	98℃	47℃	72℃	4℃
	1min	15s	15s	20s	-∞

TABLE 5 digestion system

Total volume	50μL
		Buffer	25μL
NdeI	2μL
		Xhol	2μL
Form panel	2μL
		dd H2O	19μL
Conditions of enzyme digestion	37℃，30min

TABLE 6 ligation reaction System

Total volume	10μL
		T4 DNA ligase	1μL
10*Buffer	1μL
		Carrier	1μL
Target gene	7μL

The ligation product pFastBac1-MIC1 transfer vector is transformed into Trans1-T1 competent cells, 200 mu L of bacterial liquid is taken to coat Amp⁺Culturing in a constant temperature incubator at 37 deg.C for 12 hr, and selecting single colony with regular shape from LB agar plate to Amp⁺The resistant LB liquid medium was cultured at 37 ℃ for 12 hours. After the PCR identification of the bacterial liquid, the positive bacterial liquid is sent to the company Limited in the biological engineering (Shanghai) to be sequenced, plasmids of positive bacterial colonies with correct sequencing are extracted, and the plasmids are verified by HindIII and XbaI double enzyme digestion, and two target bands which are consistent with the expectation are obtained as the result, as shown in figure 3. The pFastBac1-MIC1 transfer vector construction is shown to be successful.

Step three, constructing and identifying a recombinant baculovirus shuttle vector rebamid-MIC 1

Transforming the reaction product pFastBac1-MIC1 transfer vector in the second step into DH10Bac competent cells, coating the cells on an X-gal three-resistant agar plate, culturing for 48-72h at the constant temperature of 37 ℃, selecting white clone bacteria containing recombinant baculovirus plasmids to culture in a three-resistant LB liquid culture medium through blue-white spot screening, and extracting a recombinant baculovirus shuttle vector rebamid-MIC 1 by using a baculovirus shuttle vector bacmid small-amount extraction kit;

the recombinant baculovirus plasmid DNA was then analyzed by PCR using Bac to Bac baculovirus vector universal primers as shown in table 7, resulting in an amplification product of about 300bp for the recombinant baculovirus plasmid DNA extracted from the blue colony and 4600bp for the recombinant baculovirus plasmid DNA extracted from the white colony, as shown in fig. 4.

TABLE 7 PCR reaction System for recombinant baculovirus plasmid DNA

Total volume	50μL
		Primer Star MIX	25μL
pUC/M13-F	1μL
		pUC/M13-R	1μL
Recombinant baculovirus plasmid DNA	1μL
		dd H2O	22μL

Step four, obtaining the recombinant baculovirus strain

And adding 2 mu L of the recombinant baculovirus shuttle vector rebamid-MIC 1 prepared in the step three and 5 mu L of liposome into 100 mu L of SF900 II serum-free medium, gently mixing, and standing at room temperature for 5 min. The mixture was transfected with Sf9 insect cells in log phase growth, and a blank control group was set. After culturing at 27 ℃ for 72h, the supernatant culture was collected, which was the first generation recombinant baculovirus strain. And after collecting the supernatant culture solution, continuously carrying out three-time cell subculture to obtain the high-titer recombinant baculovirus strain.

Example 2 expression and characterization of MIC1 recombinant protein in Sf9 cells

1. Detection of ReBacmid-MIC1 transfection of Sf9 insect cells

The baculovirus genomic DNA in the supernatant of the three-cell subculture obtained in example 1 was extracted by a genomic DNA extraction kit and verified by PCR using pUC/M13-F/R-specific primers, and the results are shown in FIG. 5. PCR confirmed that rebamid-MIC 1 successfully transfected Sf9 insect cells.

Analysis of MIC1 fusion proteins

2.1 Indirect immunofluorescence detection

Sf9 insect cells 1 x 10⁶Spreading 12-well plates, culturing for 72H after infection by the recombinant baculovirus strain, carefully discarding the upper layer culture solution, washing with cold PBS three times, air-drying the cells for 15-30s, washing with 4% paraformaldehyde at room temperature for 15min, washing with cold PBS three times, then sealing with sealing solution (PBST containing 1% BSA and 22.52mg/ml glycine) at 37 ℃ for 1H, then using mouse-derived MIC1 monoclonal antibody as a primary antibody (PBST containing 1% BSA as a diluent), incubating at 37 ℃ for 1H, washing PBST three times, air-drying for 3min, adding ProLong Gold antibody mountain DAPI containing DAPI and an anti-fluorescence quencher as a secondary antibody (PBST containing 1% BSA as a diluent), incubating at 37 ℃ for 1H, washing PBST three times, washing with PBS three times, and observing by using a positive fluorescence microscope. As a result, it was found that the surfaces of the cells in the infected group were able to detect significant green fluorescence as shown in FIGS. 6, 7 and 8, while the control group showed no fluorescence as shown in FIGS. 9, 10 and 11, indicating that the recombinant rod-shaped cells were obtainedThe MIC1 recombinant protein was successfully expressed during infection of Sf9 cells by the virus.

2.2 Western blot detection of MIC1 recombinant protein

Sf9 insect cells 1 x 10⁶Spreading on a 12-hole plate, culturing for 72h after infection by the recombinant baculovirus strain, carefully removing the upper culture solution, washing with PBS for three times, cracking cells by using cell lysate, centrifuging at 12000rpm for 10min, taking the supernatant, adding 5 xSDS-PAGE loading buffer, boiling for 13min, cooling the sample to room temperature, and performing SDS-PAGE and Western blot analysis. The target protein was identified using PBST containing 1% BSA and 22.52mg/mL glycine as blocking solution, Anti-His mouse monoclonal antibody as primary antibody, and HRP-labeled goat Anti-mouse IgG (H + L) as secondary antibody. The result shows that the supernatant of cell lysate infected with recombinant baculovirus can have a specific band at 60kDa as shown in FIG. 12.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Shenyang agriculture university

<120> method for preparing baculovirus capable of soluble expressing Toxoplasma gondii MIC1 recombinant protein

<130> GG21939746A

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3955

<212> DNA

<213> Artificial Sequence

<400> 1

cttgaccatt tcaatttaga gcgcaatagt tgtagatgat cgtgttgttg tgaccagcgt 60

ttatcgattg tcagaaggcc tacgataagc gttaaggcag attctgcttg cttagggagc 120

ttaatattcg tcgaaacgat ctgaatttat agatgtcgtg aactcattta ggataagcgc 180

gcacagatag tgagcatttc tgttctcgga acttcgcctc ggtcttcttg gcaaaaaagg 240

tgtcccctct caaaatgtac tgtgtgcctg tggtggtgtt tctgtcctgg cgtcgttgtt 300

tttgaagtga gacatcaaat ttcaccgcct ttgattacgg cagaacagtc agatggtcac 360

agatgcgaca gggagtcagt gataccggtt tctgataata tttccgatgg tttttcgcat 420

gtgtactgct ggcgcgtact gaaccacctc cacacctgca ctatgtggac gttgtgcagg 480

ctcactcgct agaacgcgta acgatgaaag cagactccta atgaaaagtt tcaggtcatt 540

tcactgaaga acacgcgatt tctagtaagc agccagcgtc acatattagc gtgtcaacca 600

ccggagtaca gactgatcgt tcagtggaag acggggatta ctaaacgaaa gcaatacaga 660

gcaggagata aaagacagtc aattttgcca cgccgtctca ttcacccctt tgtatcggaa 720

tgtagtggtg ctcaccagct gtaccttcag tcactttttc tctgatacat ggtgactaaa 780

aaaggaggcc gtgtcggtgc tgctcagtaa ttgtccgccc ttgaacggtg ggtcgaaaaa 840

tccccgaaac cgtattcaca gaaagaaaaa ggggtacctt cgttcaaagt gttttccggt 900

ggtgcagctt cgggtttttt gagaaaacga agacaattaa tttgaatccg agtaaaggtc 960

acagcttaaa agagagaagg tgcacggttt gcggcgacct gtgatactgt ttaccgggag 1020

tgtccccatc tagtgtcgga actgctctac ctcacgtcat ccgtggaaat gacgttttct 1080

tccgtcactg cacatcttta cggctaccaa aacaccgttt actaatgcgc gctataaaga 1140

atcggagtct ggtagacgac tgcgtgcagt agtatctgaa aaatcactgt tctaagtact 1200

gtaggatttg acaatcgaga cggtcccgtt ccacctcacc tgatctcgaa acttacttac 1260

agtgcactac agtgagtgaa ccaaccaagt ggaggagcca ccggaagtcg gcgaaaataa 1320

cctagtacac atgcgttttc aacatttttc agagccttct acacgtcaac ctgaaagcgg 1380

gtgccgcgtc gctaccgttt cctgtggcgt ctctagtgcg acatccgaag taacagtaac 1440

gtccggcatg gaacgccgac gcgggtgttc cagtcgcctg gctccttcta ctcgcacttc 1500

gatgttacgt tccttattgg tgcgacgcgg ttctcgtgtt gctagacgtc gcaccggctg 1560

aaagctgtag aaaatttagt tattttcctg tcagctagct tgcaggagtg cgtttttgtg 1620

tgttggtttc gtctcacatg gctgctgatc tgttgatgca gctgtgtaca cgtgcctcga 1680

ttctgtagtt gacctagaac ggatttgcaa agatgggcca ggcgttgttt ctcaccgttc 1740

tattgccggt gttatttggc gttgggccag aagcatatgg agaagcgtcg cattctcatt 1800

cgccggcatc gggacgttat atacaacaga tgcttgacca acgctgccaa gagattgctg 1860

cagaactctg ccaaggcgga cttcgtaaaa tgtgtgtgcc ctctagccgg atagtagctc 1920

gaaacgccgt gggcattact catcaaaata cacttgaatg gagatgcttt gatacagcct 1980

ctttgctgga gagcaatcaa gaaaacaacg gtgttaattg cgtggacgac tgtggccaca 2040

cgataccgtg tcctggcggc gtacaccggc aaaacagtaa tcacgcaacg cgccatgaga 2100

tactgtccaa attggtcgaa gaaggagtac aacggttctg cagtccttat caagcatctg 2160

ccaacaagta ctgtaacgac aaatttccag ggaccattgc gaggaggtcg aagggcttcg 2220

gaaacaatgt cgaggttgcg tggaggtgtt acgagaaggc cagcttgctg tactcggttt 2280

atgctgagtg tgcgagcaac tgcggaacaa cgtggtactg ccctggagga cgacgaggga 2340

cgtcgacaga actagacaag cggcattata cagaagagga aggaattcgc caggcaatcg 2400

gatccgtcga cagcccatgt tctgaagttg aagtctgcct accgaaggat gagaatcccc 2460

cggtgtgttt agatgaaagt ggccagattt cacgaactgg tggtgggcca ccgtcacaac 2520

cgcctgagat gcaacagccc gccgatcgtt cggacgagag aggtggcggt aaggaacagt 2580

cgcctggagg agaagctcag ccggaccatc caacgaaggg tggtaacata gacctgcctg 2640

agaaatcaac atctcccgag aagacgccga aaaccgagat ccatggtgac agcacgaaag 2700

cgacgctcga agaggggcag caactaacgc tcacgtttat ctccactaaa ctggatgttg 2760

ctgtaggctc gtgtcattca ctcgtcgcga atttccttga tggatttttg aagtttcaga 2820

cgggctcaaa ttcggcgttc gatgtggtag aagtggaaga gccagcagga cccgcagtgc 2880

ttacgatagg tctgggacac aaaggccgtc tcgctgttgt cctcgactac accaggctca 2940

atgctgcttt aggatcagct gcttacgtgg tcgaagattc tggatgcagc tcaagtgaag 3000

aggttagttt ccaaggagtg ggtagtggag cgacgctcgt ggtgacgacg cttggcgaga 3060

gtcctacggc cgtctctgct tgatttatag tactctttgg agcatgcttg tggaggaacg 3120

ggacaatctc ggcaaaatca ggatgaagtt tgtgagatac agatcgttcc tgaacagtgg 3180

aagatgcgtc actattacac ctatatgcgt cctggttctt gtagagttgg agttcttgca 3240

ggtgtaatga ctatgacata cggatataac ttcatacggg gaactgtggc tttctcgaga 3300

ttccgccctc agtgtttctg ttgcatgccg tcccaccgta tttcggggcc ttgtattgaa 3360

tctactttct gaggttcttg cttatctctt taatgcagca tgttgcccga attcttgtta 3420

cgcggtcaga tgtttcttga gtagtgaatc aaaatgtatt atggtgtaat cctgtcagtt 3480

ttatacgtat tgtcatacgt ccacgcatct cacgtacggg cgcgaacgca gcaagtgacg 3540

agagatcatc ccactcgttt ggtgacgctg caaaatacaa gtgtattata cggtcagtcg 3600

gctctacaac attcaaaacg agttgtctcg cttcaaccac aaagcgccac actacgcaga 3660

ggaccgacgg aaggcgttaa tgcttgtcgg cgatagtcat gtcctgttta gtcgtcaaat 3720

tgcttctccc caccgggcga atcaggagtt actcgctatg tgaatagttg gagatagtgt 3780

ttcgtgagta gtaaacgggc tgttcaatgc aggtaaattc tatagccggg tcaagctcaa 3840

gcttggtggt acaacaacat ctctatgtaa aagttatttg atacgagatg ccgcgatcac 3900

atttttggag agatctgcaa catgggactt ctggtagaag gcaataaata actgc 3955

<210> 2

<211> 432

<212> PRT

<213> Artificial Sequence

<400> 2

Ala Ser His Ser His Ser Pro Ala Ser Gly Arg Tyr Ile Gln Gln Met

1 5 10 15

Leu Asp Gln Arg Cys Gln Glu Ile Ala Ala Glu Leu Cys Gln Gly Gly

20 25 30

Leu Arg Lys Met Cys Val Pro Ser Ser Arg Ile Val Ala Arg Asn Ala

35 40 45

Val Gly Ile Thr His Gln Asn Thr Leu Glu Trp Arg Cys Phe Asp Thr

50 55 60

Ala Ser Leu Leu Glu Ser Asn Gln Glu Asn Asn Gly Val Asn Cys Val

65 70 75 80

Asp Asp Cys Gly His Thr Ile Pro Cys Pro Gly Gly Val His Arg Gln

85 90 95

Asn Ser Asn His Ala Thr Arg His Glu Ile Leu Ser Lys Leu Val Glu

100 105 110

Glu Gly Val Gln Arg Phe Cys Ser Pro Tyr Gln Ala Ser Ala Asn Lys

115 120 125

Tyr Cys Asn Asp Lys Phe Pro Gly Thr Ile Ala Arg Arg Ser Lys Gly

130 135 140

Phe Gly Asn Asn Val Glu Val Ala Trp Arg Cys Tyr Glu Lys Ala Ser

145 150 155 160

Leu Leu Tyr Ser Val Tyr Ala Glu Cys Ala Ser Asn Cys Gly Thr Thr

165 170 175

Trp Tyr Cys Pro Gly Gly Arg Arg Gly Thr Ser Thr Glu Leu Asp Lys

180 185 190

Arg His Tyr Thr Glu Glu Glu Gly Ile Arg Gln Ala Ile Gly Ser Val

195 200 205

Asp Ser Pro Cys Ser Glu Val Glu Val Cys Leu Pro Lys Asp Glu Asn

210 215 220

Pro Pro Val Cys Leu Asp Glu Ser Gly Gln Ile Ser Arg Thr Gly Gly

225 230 235 240

Gly Pro Pro Ser Gln Pro Pro Glu Met Gln Gln Pro Ala Asp Arg Ser

245 250 255

Asp Glu Arg Gly Gly Gly Lys Glu Gln Ser Pro Gly Gly Glu Ala Gln

260 265 270

Pro Asp His Pro Thr Lys Gly Gly Asn Ile Asp Leu Pro Glu Lys Ser

275 280 285

Thr Ser Pro Glu Lys Thr Pro Lys Thr Glu Ile His Gly Asp Ser Thr

290 295 300

Lys Ala Thr Leu Glu Glu Gly Gln Gln Leu Thr Leu Thr Phe Ile Ser

305 310 315 320

Thr Lys Leu Asp Val Ala Val Gly Ser Cys His Ser Leu Val Ala Asn

325 330 335

Phe Leu Asp Gly Phe Leu Lys Phe Gln Thr Gly Ser Asn Ser Ala Phe

340 345 350

Asp Val Val Glu Val Glu Glu Pro Ala Gly Pro Ala Val Leu Thr Ile

355 360 365

Gly Leu Gly His Lys Gly Arg Leu Ala Val Val Leu Asp Tyr Thr Arg

370 375 380

Leu Asn Ala Ala Leu Gly Ser Ala Ala Tyr Val Val Glu Asp Ser Gly

385 390 395 400

Cys Ser Ser Ser Glu Glu Val Ser Phe Gln Gly Val Gly Ser Gly Ala

405 410 415

Thr Leu Val Val Thr Thr Leu Gly Glu Ser Pro Thr Ala Val Ser Ala

420 425 430

Claims (10)

1. The preparation method of the baculovirus capable of soluble expressing the Toxoplasma gondii MIC1 recombinant protein is characterized by comprising the following steps:

s1, analyzing the gene sequence and protein of toxoplasma gondii microwin TGME49_291890, cutting off the 1-72 bit sequence of the coding signal peptide to obtain the sequence shown in SEQ1 as the target gene;

s2, designing primers MIC1-NdeI-5 and MIC1-XhoI-3 with enzyme cutting sites for MIC 1;

s3, using cDNA as a template, amplifying a target gene fragment by using the primer in the step S2, carrying out NdeI and XhoI double-enzyme digestion to obtain a target fragment with an enzyme digestion site, carrying out NdeI and XhoI double-enzyme digestion on a pET22b vector, respectively purifying and recovering the target fragment after enzyme digestion and a linearized vector, and connecting the target fragment and the linearized vector through ligase overnight to obtain a connection product pET22b-MIC 1;

s4, designing primers MIC1-XbaI-5 and MIC1-Hind III-5 with enzyme cutting sites for pET22b-MIC 1;

s5, using pET22b-MIC1 as a template, adopting the primer in the step S4 to amplify a target fragment, performing double enzyme digestion by Hind III and XbaI to obtain the target fragment with an enzyme digestion site, performing double enzyme digestion by Hind III and XbaI to obtain an insect cell expression vector pFastBac1, respectively purifying and recovering the target fragment after enzyme digestion and a linearized vector, and performing a connection reaction on the target fragment and the linearized vector through ligase to obtain a connection product pFastBac1-MIC1 transfer vector;

s6, transforming the connecting product pFastBac1-MIC1 transfer vector to a DH10Bac competent cell, and obtaining a recombinant baculovirus shuttle vector rebamid-MIC 1 through resistant blue-white spot screening and separation;

s7, transfecting the recombinant baculovirus shuttle vector rebamid-MIC 1 to Sf9 insect cells, and carrying out three-time cell subculture to obtain the recombinant baculovirus.

2. The method for preparing the baculovirus with the solubility expressing the Toxoplasma gondii MIC1 recombinant protein as claimed in claim 1, wherein the baculovirus comprises: the primer sequence in step S2 is:

MIC1-NdeI-5：5'-CCTTCATATGGCGTCGCATTCTCAT-3', the restriction sites NdeI are underlined;

MIC1-XhoI-3：5'-TAACCTCGAGAGCAGAGACGGCCG-3', the cleavage site XhoI is underlined.

3. The method for preparing the baculovirus with the solubility expressing the Toxoplasma gondii MIC1 recombinant protein as claimed in claim 1, wherein the baculovirus comprises: the amplification in the step S3 adopts PCR amplification; the PCR amplification reaction parameters are as follows: pre-denaturation at 98 ℃ for 1min, denaturation at 98 ℃ for 10s, annealing at 47 ℃ for 15s, and extension at 72 ℃ for 10s, after 10 cycles; denaturation at 98 ℃ for 10s, annealing at 69 ℃ for 15s, extension at 72 ℃ for 10s, re-extension at 4 ℃ and 25 cycles.

4. The method for preparing the baculovirus with the solubility expressing the Toxoplasma gondii MIC1 recombinant protein as claimed in claim 1, wherein the baculovirus comprises: the temperature of the overnight ligation in step S3 was 16 ℃.

5. The method for preparing the baculovirus with the solubility expressing the Toxoplasma gondii MIC1 recombinant protein as claimed in claim 1, wherein the baculovirus comprises: the temperature of the enzyme digestion in the step S3 is 37 ℃, and the time is 30 min.

6. The method for preparing the baculovirus with the solubility expressing the Toxoplasma gondii MIC1 recombinant protein as claimed in claim 1, wherein the baculovirus comprises: step S3 is described in the amplification process reactant buffer solution, NdeI, XhoI, template, dd H₂The volume ratio of O is 25:2:2:2: 19; the ligase in the ligation reaction process is T4 DNA, wherein the volume ratio of reactant T4 DNA ligase, 10 × Buffer, vector and target gene is 1:1:1: 7.

7. The method for preparing the baculovirus with the solubility expressing the Toxoplasma gondii MIC1 recombinant protein as claimed in claim 1, wherein the baculovirus comprises: the primer sequence in step S4 is:

MIC1-XbaI-5：5'-TTAATCTAGAATGGCGTCGCATTCTC-3', the restriction site XbaI is underlined;

MIC1-HindⅢ-5：5'-TTGCAAGCTTTCAGTGGTGGTGG-3', the restriction site Hind III is underlined.

8. The method for preparing the baculovirus with the solubility expressing the Toxoplasma gondii MIC1 recombinant protein as claimed in claim 1, wherein the baculovirus comprises: in the step S5, PCR amplification is adopted for amplification; PCR amplification reaction parameters: pre-denaturation at 98 ℃ for 1min, denaturation at 98 ℃ for 15s, annealing at 47 ℃ for 15s, extension at 72 ℃ for 20s, re-extension at 4 ℃ and 35 cycles.

9. The method for preparing the baculovirus with the solubility expressing the Toxoplasma gondii MIC1 recombinant protein as claimed in claim 1, wherein the baculovirus comprises: step S5 is the amplification process reactant buffer solution, NdeI, XhoI, template, dd H₂The volume ratio of O is 25:2:2:2: 19; the ligase in the ligation reaction process is T4 DNA, wherein the reactant T4 DNA is ligatedThe volume ratio of the enzyme to the 10-star Buffer to the vector to the target gene is 1:1:1: 7.

10. The method for preparing a baculovirus soluble expressing toxoplasma MIC1 recombinant protein according to any one of claims 1 to 9, wherein: the MIC1 recombinant protein expressed by the recombinant baculovirus in insect cells has solubility and antigenicity.

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