Optimized PCV2d ORF2 gene and preparation method of virus-like particles
阅读说明：本技术 一种优化的PCV2d ORF2基因及病毒样颗粒的制备方法 (Optimized PCV2d ORF2 gene and preparation method of virus-like particles ) 是由 缪德年 夏叶 李春华 郑琳琳 王宏华 黄冬 薄宗义 廖学文 于 2019-03-04 设计创作，主要内容包括：本发明属于分子生物学领域,公开了一种优化的PCV2d ORF2基因及其病毒样颗粒的制备方法。本发明对编码猪圆环病毒2d亚型Cap蛋白的编码基因序列优化后,构建表达工程菌表达系统进行Cap蛋白的表达。在适合的表达条件下,该表达菌可大量表达可溶且具有活性的猪圆环病毒2d亚型Cap蛋白,利用单克隆抗体柱对表达蛋白进行纯化,得到高纯度高浓度的Cap蛋白,且该蛋白可形成病毒样颗粒。本发明实现了猪圆环病毒2d亚型全长Cap蛋白在原核表达系统中的高效可溶表达,操作简单,成本低廉,制备的单克隆抗体柱能反复使用,纯化得到的Cap蛋白具有高浓度和高纯度,且能自发形成病毒样颗粒,免疫原性好,适合工业化应用。(The invention belongs to the field of molecular biology, and discloses an optimized PCV2d ORF2 gene and a preparation method of virus-like particles thereof. After the coding gene sequence of the 2d subtype Cap protein of the porcine circovirus is optimized, an expression engineering bacteria expression system is constructed to express the Cap protein. Under a proper expression condition, the expression strain can express a large amount of soluble and active porcine circovirus 2d subtype Cap protein, the expression protein is purified by using a monoclonal antibody column to obtain the Cap protein with high purity and high concentration, and the protein can form virus-like particles. The invention realizes the efficient soluble expression of the porcine circovirus 2d subtype full-length Cap protein in a prokaryotic expression system, has simple operation and low cost, the prepared monoclonal antibody column can be repeatedly used, the Cap protein obtained by purification has high concentration and high purity, can spontaneously form virus-like particles, has good immunogenicity and is suitable for industrial application.)
1. A DNA encoding a porcine circovirus subtype 2d Cap protein and having a similarity of more than 80% to the sequence of SEQ ID NO 2.
2. The DNA of claim 1, wherein the DNA has the sequence shown in SEQ ID NO 2.
3. A plasmid which is an expression vector containing the DNA according to claim 1.
4. A protein having a nucleic acid coding sequence with greater than 80% similarity to the sequence of SEQ ID NO 2.
5. A cell comprising the DNA of claim 1.
6. A monoclonal antibody capable of specifically binding to the protein of claim 4.
7. The monoclonal antibody according to claim 6, which is obtained by immunizing an animal with the protein according to claim 4, producing a hybridoma and performing monoclonal culture.
8. A monoclonal antibody column conjugated with the monoclonal antibody of claim 6.
9. A method for producing a Cap protein, characterized in that the DNA according to claim 1 is ligated to an expression vector to express a protein.
10. The use of the DNA of claim 1, wherein the virus-like particles are prepared by an E.coli expression system, comprising the steps of:
(1) inserting the DNA of claim 1 into an Escherichia coli expression vector to form a recombinant expression vector, and then transforming the recombinant expression vector into Escherichia coli expression host bacteria to construct expression engineering bacteria;
(2) carrying out secretory expression on the recombinant escherichia coli expression engineering bacteria to obtain soluble Cap protein;
(3) and purifying the expressed Cap protein by using a monoclonal antibody column, wherein the obtained purified protein can form virus-like particles.
11. The use of claim 10, wherein the e.coli expression vector is pET28a (+), pET30a (+) or pET32a (+).
12. The use according to claim 10, wherein the E.coli expression host cell is E.coli BL21(DE3), E.coli BL21(DE3) pLySs or E.coli Rosetta (DE 3).
13. The use of claim 10, wherein the secretion expression of the recombinant escherichia coli expression engineering bacteria is that overnight cultured expression engineering bacteria are taken and inoculated in LB liquid culture medium containing kanamycin, and shake culture is carried out at 37 ℃ until OD600 is 0.6-0.8; adding IPTG with final concentration of 0.0001-0.0005mol/L, and inducing and culturing at 37 deg.C for 5-7 h.
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a preparation method for obtaining soluble PCV2d Cap protein and virus-like particles by using an optimized PCV2d ORF2 gene and a monoclonal antibody column for purification.
Background
Porcine circovirus type 2(PCV2) is the main pathogen of porcine circovirus-related diseases causing postweaning multisystemic wasting syndrome, porcine dermatitis nephrotic syndrome, proliferative necrotizing pneumonia and the like. Currently, PCV2 is ubiquitous in swine herds in China and even the world, causing serious economic losses to the swine industry. Once a pig is infected with the circovirus disease, no effective medicine is used for treatment, so vaccine immunization is a main way for preventing the disease. PCV2 has 11 Open Reading Frames (ORFs) in total, of which ORFs 1 and 2 are the two most prominent reading frames. Research shows that ORF2 is the main structural protein and capsid protein (Cap) of the encoded virus, and the epitope on the Cap protein has immunogenicity and is the basis of vaccine development.
The PCV2 vaccine on the market is mainly inactivated vaccine and subunit vaccine. The inactivated vaccine has poor immune effect, can achieve the prevention effect only by strengthening immunity for many times, and can cause the safety problem because the inactivation process is incomplete. Subunit vaccines are all produced by adopting a eukaryotic expression system, however, because the expression level of the Cap protein encoded by ORF2 in the eukaryotic expression system is extremely low, even if a high-expression baculovirus-insect cell expression system is utilized, the expression level is usually difficult to exceed 50-100mg/L (Pumpens P, Grens E.HBV core particles as a carrier for B cells/Tcell epitopes. Intervirology,2001,44: 98-114.), and therefore, the yield is low, the cost is high, and the vaccines are difficult to be used in production in farms.
In order to increase the yield, the expression of Cap protein is carried out by using an E.coli expression system, but usually only insoluble form of denatured inclusion body protein can be obtained, and soluble protein can be obtained only by protein renaturation, and the method of denaturation followed by renaturation has certain loss on protein yield and protein activity (Lizhong, Wang Fengqi, Wujiamin, etc.. A preparation method of porcine circovirus type 2 virus-like particles and virus-like particles obtained by the method: China, CN201710439243.9[ P ], 2017.10.13.).
PCV2 can be classified into PCV2a, PCV2b, PCV2c, PCV2d, and PCV2 e. The epidemic strain of PCV2 before 2004 is mainly PCV2a subtype, and after 2004, the epidemic trend gradually changes to PCV2b subtype, and two new subtypes, PCV2d and PCV2e, continuously appear. After 2014, PCV2D subtype gradually replaced PCV2b subtype to become dominant serotype (serological survey of porcine circovirus disease in Xuni. Shandong province and genetic variation analysis of PCV2 isolate [ D)]Thailan, university of shandong agriculture, 2014; xiao CT, Halbur PG, Opriessnig T.Global molecular genetic analysis of pore circular type 2(PCV2) sequences constants the present of four main PCV2 genes and derivatives a Rapid increment of PCV2d [ J]J Gen Virol, 2015,96: 1830-. Current commercial vaccines, e.g. of the company Merriya, FranceOf Blringger corporation, USA
Virus Like Particles (VLPs) are protein multimeric complexes with similar spatial structure and immunogenicity to native viruses, can be recognized by TLRs and PRRs of host cells, thereby activating innate immune mechanisms, and can induce strong B cell responses due to their regular surface structure; and is beneficial to the antigen presentation of the antigen presenting cells, thereby having strong immunogenicity. VLPs, however, do not contain viral genetic material and are therefore non-infectious, stable, not prone to inactivation, and can induce humoral, cellular and mucosal immunity. Based on the advantages of VLPs, the VLPs can enhance the immune effect of the vaccine, reduce the immune times, reduce the cost, improve the production performance and have wide development prospect. However, Cap proteins expressed using E.coli or yeast expression systems often fail to spontaneously assemble into VLPs (Masuda A1, Lee JM1, Miyata T, Purification and characterization of immunogenic recombinant viruses-like proteins of a bacterial circular type 2expressed in a plasmid pure plant, J GenVirol, 2018 May 31), and thus optimization of expression conditions and gene sequences is particularly important.
In conclusion, the prevention and control of the circovirus disease mainly depends on vaccine immunization, and a new vaccine is needed for prevention and control of the new epidemic genotype PCV2d subtype. The yield of Cap protein obtained by the current eukaryotic expression system is low and the cost is high; although the Escherichia coli expression system is simple to operate, high in expression quantity and low in cost, the obtained Cap protein is generally poor in solubility, large in purification difficulty and low in VLPs formation rate.
Disclosure of Invention
The first technical problem to be solved by the invention is to obtain a soluble Cap protein coding gene.
The second technical problem to be solved by the invention is to prepare the PCV2d Cap protein with high expression and solubility.
The third technical problem to be solved by the present invention is to obtain VLPs having high purity, good activity, and capable of forming Cap proteins of uniform size.
At present, the commercial PCV2 vaccine has the following problems: the genotype that the existing vaccine can control does not cover the current popular PCV2d genotype; the commercial subunit vaccine expressed by the eukaryotic system has low yield and high cost; cap protein obtained by an Escherichia coli expression system is generally poor in solubility, large in purification difficulty and low in VLPs formation rate. In order to overcome the defects, the invention obtains the optimized ORF2 gene which is easy to be soluble and expressed and codes the Cap protein according to the current popular PCV2d genotype, and utilizes the optimized ORF2 gene and an Escherichia coli expression system to prepare the high-expression and soluble PCV2d Cap protein; the Cap protein obtained by purifying the monoclonal antibody column has high purity and good activity, and can form VLPs with uniform size.
According to the first aspect of the invention, an optimized PCV2d ORF2 gene is provided, and an expression engineering bacterium constructed by using the optimized gene can be used for soluble expression of PCV2d Cap protein under a proper induction and expression condition, and the Cap protein can form virus-like particles (VLPs).
In a second aspect of the present invention, there is provided a soluble PCV2dCap protein expressed by the expression cassette of the first aspect of the present invention.
In a third aspect of the invention, an active PCV2dCap protein is provided, the agar titer can reach 1:64, and the active Cap protein is expressed by the expression engineering bacteria.
The fourth aspect of the invention provides a monoclonal antibody of PCV2d Cap protein, and the purity of Cap protein obtained by purifying an antibody column prepared by the monoclonal antibody can reach more than 90%.
In a fifth aspect of the invention, there is provided virus-like particles (VLPs) expressed by the expression construct of the first aspect of the invention.
The invention provides a DNA, which codes 2d subtype Cap protein of porcine circovirus and has more than 80% of similarity with the sequence of SEQ ID NO 2.
Preferably, the sequence of the DNA is shown as SEQ ID NO 2.
ORF2 gene (shown as SEQ ID NO.1) in the genome of PCV2d strain is optimized according to codon preference of escherichia coli to obtain an optimized sequence (shown as SEQ ID NO. 2. due to the degeneracy of codons, even a DNA sequence with 80% homology with SEQ ID NO 1 or SEQ ID NO 2 can obtain the same protein.
The present invention provides a plasmid which is an expression vector containing the above DNA.
The invention also provides a corresponding protein, and the nucleic acid coding sequence of the protein has more than 80% of similarity with the sequence of SEQ ID NO 2.
The invention also provides a cell containing the DNA.
The present invention also provides a monoclonal antibody capable of specifically binding to the protein encoded by the above DNA. For example, an antibody can be obtained by immunizing an animal with the above-described protein, preparing a hybridoma, and performing monoclonal culture.
Accordingly, the present invention provides a monoclonal antibody column, which is coupled with the monoclonal antibody and can be used for purifying the protein.
In another preferred embodiment, the ORF2 sequence is a full-length sequence.
In another preferred embodiment, the ORF2 sequence is optimized according to E.coli codon preference.
In another preferred embodiment, the expression cassette comprises the following elements operably linked 5 'to 3': a start codon, the optimized ORF2 sequence, and a stop codon.
In another preferred embodiment, the optimized gene sequence of PCV2d ORF2 is shown as SEQ ID NO.2 (containing a start codon and a stop codon).
In another preferred embodiment, the expression vector has a pET30a vector as a backbone.
In another preferred embodiment, the expression engineering bacterium is Escherichia coli, preferably Rosetta (DE 3).
In another preferred example, the virus-like particle is formed by Cap protein expressed by PCV2d ORF2 gene.
The invention provides a preparation method of Cap protein, which connects DNA with a sequence of SEQ ID NO 2 with an expression vector to express protein.
In the present invention, the virus-like particles can be prepared by an E.coli expression system, and the preparation steps are as follows:
inserting DNA with 80% homology with SEQ ID NO 1 sequence into an escherichia coli expression vector to form a recombinant expression vector, and then transforming the recombinant expression vector into escherichia coli expression host bacteria to construct expression engineering bacteria;
carrying out secretory expression on the recombinant escherichia coli expression engineering bacteria to obtain soluble Cap protein;
and purifying the expressed Cap protein by using a monoclonal antibody column, wherein the obtained purified protein can form virus-like particles.
In the invention, the Escherichia coli expression vector can be pET28a (+), pET30a (+) or pET32a (+).
In the present invention, the E.coli expression host cell may be E.coli BL21(DE3), E.coli BL21(DE3) pLySs or E.coli Rosetta (DE 3).
In the invention, the secretion expression of the recombinant escherichia coli expression engineering bacteria refers to that overnight cultured expression engineering bacteria are taken and inoculated in an LB liquid culture medium containing kanamycin, and shake culture is carried out at 37 ℃ until OD600 is 0.6-0.8; adding IPTG with final concentration of 0.0001-0.0005mol/L, and inducing and culturing at 37 deg.C for 5-7 h.
Specifically, the invention comprises the following technical scheme:
synthesis of target gene and construction of recombinant expression plasmid: according to the ORF2 gene (the sequence is shown as SEQ ID NO.1) in the genome of the PCV2d strain and the codon preference of escherichia coli, an optimized sequence (the sequence is shown as SEQ ID NO.2) is obtained through optimization, after enzyme cutting sites are introduced, the optimized sequence is cloned to expression vectors pET28a (+), pET30a (+), pET32a (+), and preferably pET30a (+), and a recombinant expression plasmid carrying PCV2d ORF2 is obtained.
Construction of recombinant expression bacteria: the recombinant expression plasmid is transformed into Escherichia coli expression host bacteria E.coli BL21(DE3), E.coli BL21(DE3) pLySs and E.coli Rosetta (DE3), preferably E.coli Rosetta (DE3), to obtain the expression engineering bacteria.
Prokaryotic expression of Cap protein: taking overnight-cultured expression engineering bacteria, inoculating the expression engineering bacteria into an LB liquid culture medium containing kanamycin, and carrying out shake culture at 37 ℃ until OD600 is 0.6-0.8; adding IPTG with final concentration of 0.0001-0.0005mol/L, and inducing and culturing at 37 deg.C for 5-7 h.
Ultrasonic cracking of recombinant expression bacteria: and after collecting the bacteria, washing the bacteria once by PBS, collecting the bacteria again, suspending the bacteria by the PBS, performing ultrasonic pyrolysis in an ice water bath, and collecting supernatant, namely the soluble PCV2d-Cap protein.
Crude purification of Cap protein: adding saturated ammonium sulfate solution into the crude protein solution, standing at 4 deg.C, centrifuging, collecting supernatant, repeating the above steps, centrifuging, collecting precipitate, and resuspending with PBS; obtaining PCV2d-Cap crude pure protein by using a discontinuous sucrose density gradient centrifugation method.
Preparing and purifying Cap protein monoclonal antibody: the steps comprise animal immunization, establishment and cloning of hybridoma cell strains and preparation and purification of ascites.
Animal immunization: the Cap protein mixed with the adjuvant and fully emulsified is used as immunogen to immunize mice, and the mice with serum antibody titer more than 1:104 are taken for cell fusion after the three-immunization. 3 days before fusion, inoculating Cap protein into abdominal cavity to perform super-strong immunity;
establishment and cloning of hybridoma cell lines: after 3 days of boosting immunity, aseptically taking mouse splenocytes and mouse myeloma cells (SP2/0) for fusion culture, screening positive through IFA, cloning and culturing until the positive rate of antibody secretion is more than 95%, and then carrying out expanded culture;
preparing and purifying ascites: inoculating the hybridoma cells to the desensitized mouse abdominal cavity, collecting ascites, and obtaining a purified ascites supernatant as the monoclonal antibody.
Monoclonal antibody specificity analysis: the specificity of the prepared monoclonal antibody is identified by indirect ELISA and Western-Blot.
Preparation of monoclonal antibody column: and (3) fully mixing the monoclonal antibody displacement liquid and the medium, adding a proper amount of sealing liquid for sealing, and finally washing with a cleaning liquid and PBS to obtain the monoclonal antibody column.
Fine purification of Cap protein: placing the monoclonal antibody column into a binding buffer solution, balancing the column by using the binding buffer solution with 5 times of volume, loading the Cap crude protein into the monoclonal antibody column, eluting to obtain Cap protamine, and dialyzing and desalting.
Detection of virus-like particles: and (3) dripping a trace amount of the purified Cap protein on a copper net, dyeing, drying, and observing the formation condition of virus-like particles under a transmission electron microscope.
In the subsequent embodiments of the invention, it can be seen that the invention has at least the following advantages:
the Escherichia coli expression system is simple to operate, short in period and mature in technology. Only 1 week is needed from the construction of engineering bacteria to the prokaryotic expression of PCV2d Cap protein by an escherichia coli expression system; eukaryotic expression generally takes 4-6 weeks from the construction of the vector to the expression of the protein. Therefore, the protein expression by using the escherichia coli expression system greatly simplifies the experimental steps and shortens the experimental period.
The escherichia coli expression system is used for carrying out the secretion expression of PCV2dCap protein, and the cost is low. The whole protein expression process does not need to culture insect cells in a large scale, only needs to culture escherichia coli, and greatly reduces the production cost.
The expression amount is high. The protein yield of prokaryotic expression is higher than that of eukaryotic expression, and the yield in unit time is greatly higher than that of eukaryotic expression due to short production period. In the subsequent embodiment, the expression quantity of PCV2dCap protein obtained by prokaryotic expression can reach more than 150mg/L culture solution, and if the industrial production is mature, the yield can be continuously improved.
Obtaining the soluble protein with activity. The protein produced by the invention is soluble protein, renaturation is not needed, and the loss of protein amount in the renaturation process is reduced; has good protein activity and can reach higher agar titer.
The PCV2d Cap protein purified by the monoclonal antibody column has high purity which can reach more than 90 percent.
The purified soluble protein spontaneously forms VLPs. VLPs have good immunogenicity, so that Cap proteins capable of forming VLPs can enhance the immune effect of vaccines and reduce the immune frequency, thereby reducing the production cost and improving the production performance.
Drawings
FIG. 1: SDS-PAGE electrophoresis chart of induced expression and solubility analysis of PCV2d Cap protein.
Wherein: m: protein molecular weight standards; 1: uninduced Cap protein supernatant; 2: uninduced Cap protein precipitation; 3: induced empty plasmid supernatant; 4: induced precipitation of empty plasmids; 5: induced Cap protein supernatant; 6: induced precipitation of Cap protein.
FIG. 2: purification of PCV2d Cap protein.
Wherein, M: protein molecular weight standards; 1: purifying the crude Cap protein; 2: and purifying the Cap protein by using a monoclonal antibody column.
FIG. 3: an electron microscope image of virus-like particles formed by PCV2d Cap protein.
Among them, electron microscopy of VLPs (67000X).
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following examples. It is to be understood that this example is intended only to illustrate the present invention and is not intended to limit the scope of the present invention, and that the specific experimental procedures not mentioned in the following examples should be carried out according to the conventional experimental procedures.