CN113549634B - Gene for coding soluble HPV58L1 protein and construction and application of recombinant plasmid thereof - Google Patents

Abstract

The invention discloses a gene for coding soluble HPV58L1 protein and construction and application of recombinant plasmid thereof. The gene HPV58L1 is obtained by optimization design on the basis of comprehensive multiple complex factors, and recombinant plasmids pUC-58L1 and pESUMO-58L are constructed. The HPV58L1 gene can obviously improve the soluble expression efficiency of the target protein, and the product has uniform property and stable performance, and the expression quantity of the target protein can meet the requirement of industrial production; the invention provides a low-cost prokaryotic expression method of soluble HPV58L1 protein, which is characterized in that the HPV58L1 protein and SUMO protein are subjected to fusion expression, and recombinant expression protein with good solubility and obvious hemagglutination activity can be obtained.

Description

Construction and application of gene encoding soluble HPV58 L1 protein and its recombinant plasmid

technical field

The invention relates to the technical field of genetic engineering, in particular to the construction and application of a gene encoding soluble HPV58 L1 protein and its recombinant plasmid.

Background technique

According to statistics, in 2018, there were 570,000 new cases of cervical cancer and 311,000 deaths in the world. It is the fourth most common cancer in women. Long-term persistent infection is an important cause of cervical cancer. Human papillomavirus (Human Papillomavirus, HPV) is a type of double-stranded small molecule DNA virus with strict species specificity. It mainly infects human skin and mucosal tissues with high diversity and species specificity. There are significant differences in the distribution of race and ethnicity. HPV 58 is one of the most common carcinogenic subtypes; globally, HPV 58 ranks sixth in the cause of cervical cancer, but it is more prevalent in East Asia, where its detection rate ranks only after HPV 16 and HPV After 18, it ranks third, accounting for 10% to 18% of all cases. In addition to cervical cancer, HPV infection is also closely related to diseases such as anogenital cancer, squamous cell carcinoma of the head and neck, and skin warts.

HPV is a small dsDNA virus with a genome size of about 8000 bp, a molecular weight of about 5×10 ⁶ Daltons, a diameter of 50-60 nm, and an icosahedral structure. Contains 8 open reading frames (Open reading frame, ORF), each encoding different proteins, according to their expression time, they are divided into: early region, late region and long control region. The early region is located downstream of the genome, encoding non-structural proteins E1, E2, E4, E5, E6 and E7 involved in virus replication and transcription. The late region is about 2500 bp in size and encodes L1 and L2 structural proteins, which together constitute the capsid of the virus. L1 protein is the main capsid protein of HPV, encoded by L1 ORF, because the conservation of L1 ORF is very high, it is used as the basis for HPV typing. L1 protein size is about 55 ~ 60 kDa. Generally, 5 L1 proteins polymerize to form a 5-mer, and 72 5-mers and a certain number of L2 proteins together form a viral capsid with T=7 20-hedral structure.

Studies have shown that L1 protein can self-assemble under suitable conditions to form virus like particles (Virus like particles, VLP). Currently, all HPV preventive vaccines approved for marketing are developed based on VLP. The development of expression systems has high production costs, high prices and limited protection subtypes, and the penetration rate in developing countries and low-income areas is low.

Contents of the invention

The purpose of the present invention is to provide a gene encoding soluble HPV58 L1 protein, its recombinant plasmid, and recombinant engineering bacteria, and construct a method for expressing HPV58 L1 protein, aiming at improving the solubility, activity and expression of HPV 58 L1 protein, and reducing its preparation cost.

In order to solve the problems of the technologies described above, the present invention adopts the following technical solutions:

Based on the transformation of the wild-type HPV58L1 gene, all the corresponding amino acids use the most frequently used nucleotide codons in Escherichia coli; and in order to avoid the high proportion of GC in the translated mRNA, the secondary structure of the mRNA has a great influence on the translation efficiency. The optimal codon frequency was corrected by avoiding some commonly used enzyme cutting sites; finally, on the basis of comprehensive consideration of multiple complex factors, a new HPV58 L1 DNA sequence was designed, such as SEQ ID NO. 1.

The synthetically optimized HPV58L1 DNA gene was loaded into the pUC57 plasmid to form the recombinant plasmid pUC-58L1; and the recombinant plasmid pESUMO-58L was constructed.

The method for constructing the recombinant plasmid expressing soluble HPV58 L1 protein comprises the steps:

(1) Design the amplification primers of the gene HPV23L1, and its forward primer includes a Bsa I restriction endonuclease site, and its reverse primer includes an Xho I restriction endonuclease site flanking the stop codon;

(2) PCR amplifying the gene HPV58L1 with the amplification primers;

(3) Take the pESUMO plasmid and the resulting PCR amplification products and perform double digestion with restriction endonucleases Bsa I and Xho I respectively, recover the double digestion products of the empty vector plasmid pE-SUMO and HPV58L1 gene, and connect them with T4 DNA The enzyme was ligated overnight at 16°C to obtain a recombinant plasmid;

(4) Transform Escherichia coli competent cells BL21 with the recombinant plasmid pESUMO-58L1 obtained in the previous step, spread it on Amp ⁺ /LB solid medium for culture, pick a single colony for bacterial liquid PCR identification, screen positive colonies, and extract positive colonies Plasmid, the correct sequencing result is the recombinant plasmid expressing soluble HPV58 L1 protein.

The sequences of the amplification primers are as follows: Primer name Restriction sites sequence 5'-3' F-58 <i>Bas I</i> TT<u><i>GGTCTC</i></u>TAGGTATGTCCGTGTGGCGTC R-58 <i>Xho I</i> CCG<u><i>CTCGAG</i></u>TTATTTTTTAACCTTTTTGCGT

The preparation method of soluble human papillomavirus 58 subtype L1 protein comprises the following steps:

(1) The recombinant plasmid pESUMO-58L1 was inoculated in Amp ⁺ /LB liquid medium and cultured until the OD ₄₅₀ value reached 0.75-0.85, and the expression was induced at the concentration of the inducer IPTG at 0.3 mmol/L and 18°C;

(2) After the induced expression is completed, collect the bacteria by centrifugation, wash and break up, and centrifuge to collect the supernatant;

(3) The supernatant was eluted and purified through a Ni affinity chromatography column to obtain SUMO-58 L1 protein.

Compared with the prior art, the main beneficial technical effects of the present invention are:

(1) The present invention optimizes the HPV58L1 gene so that it can efficiently express the target protein HPV58L1 in the E. coli host, and based on the SUMO tag expression system, it is fused and expressed in E. coli, which increases the solubility of the target protein and also makes the expressed The target protein has higher biological activity.

(2) The gene and its expression system of the present invention can significantly improve the efficiency of soluble expression of the target protein, and the obtained purified product has uniform properties and good stability; its protein expression is as high as 100 μg/ml, which can meet the requirements of industrial production.

(3) The present invention provides a low-cost method for prokaryotic expression of soluble HPV58L1 protein. In this method, HPV58L1 protein is fused with SUMO protein to obtain a recombinant expression protein with good solubility and obvious hemagglutination activity.

Description of drawings

Fig. 1 is the bacterium fluid PCR of recombinant plasmid and double-enzyme digestion identification map; Wherein,

Figure A is the bacterial liquid PCR identification of pESUMO-58L1; M. Marker; lanes 1-8. pESUMO-58L1 amplified product;

Figure B is the identification of double restriction enzyme digestion of the recombinant expression vector: M. Marker; Lane 1 is the product of double digestion of pESUMO-58L1.

Figure 2 is the preliminary expression and identification map of the recombinant SUMO-L1 protein; wherein,

Figure A is 12% SDS-PAGE identification; M: Protein Marker; 1: Uninduced pE-SUMO-L1 empty bacteria; 2: pE-SUMO-L1 expressing bacteria induced for 10 h; 3: BL21 (DE3) empty bacteria control group(NC);

Figure B is Western Blot identification; M: Protein Marker; 1: NC control; 2: SUMO-L1 recombinant protein;

Panel C is the solubility analysis. M: protein marker; 1: ultrasonic precipitation; 2: ultrasonic supernatant.

Figure 3 is a purification and identification diagram of recombinant protein expressed by Escherichia coli of HPV58L1; wherein,

Figure A is the identification result of 12% SDS-PAGE; M. Protein Marker; 1. SUMO-58L1 protein before purification; 2. SUMO-58L1 protein after purification;

Figure B is Western Blot identification. In the figure, M. Protein Marker; 1. Purified SUMO-58L1 protein.

Figure 4 is the purification of SUMO protease and the SUMO-tag excision results of the recombinant protein SUMO-58L1 protein; wherein,

Figure A is the SDS-PAGE identification of the purified SUMO protease (Ulp1); where M. Protein Marker; 1. Ulp1 after purification;

Figure B is the SDS-PAGE identification of UMO-tag excision; M. Protein Marker; 1. SUMO-58L1 protein; 2. Products after enzyme digestion; 3. Purified and concentrated HPV58L1 protein.

Figure 5 is the identification of the hemagglutination activity of the recombinant protein, wherein 1:2 to 1:1024 is the result of 2-fold dilution of the purified SUMO-58L1 protein; NC is the PBS negative control.

Fig. 6 is the ebb and flow rule and titer determination result of immune mouse serum antibody; Wherein,

Figure A shows the serum ELISA test results at 0, 7, 14, 21, 28 and 36 days after immunization;

Figure B shows the titer detection results of mouse serum 36 days after immunization.

Figure 7 is the result of indirect immunofluorescence (IFA) identification; among them,

Figure A shows 293T cells transfected with pcDNA3.1-GFP;

Figure B shows the reaction with immune serum after pcDNA3.1-HPV58 L1 transfected 293T cells;

Figure C shows the use of PBS instead of mouse serum as a blank control (BC);

Figure D shows the serum of mice immunized with PBS as negative control (NC).

Detailed ways

The specific implementation of the present invention will be described below in conjunction with the accompanying drawings and examples, but the following examples are only used to describe the present invention in detail, and do not limit the scope of the present invention in any way.

The experimental methods without specific conditions indicated in the following examples are usually carried out according to conventional conditions, such as the conditions described in ("Molecular Cloning: A Laboratory Manual", the fourth edition of the original book, 2012).

In the following examples, DNA extension and PCR amplification reagents and restriction endonucleases BsaI, EcoRI and XhoI were purchased from NEB ( New England Biolabs, Inc. ); the anti-HRP-labeled goat anti-mouse IgG used was purchased from Abcam.

In the following examples, the formulation of the bacteriostasis buffer used in the purification step is: PBS (3-morpholinopropanesulfonic acid), pH 7.4.

Embodiment 1, the construction of recombinant expression plasmid

1. Cloning of HPV58 L1 protein coding region gene

1.1 Design and synthesis of HPV58L1 gene sequence

The HPV58L1 gene sequence of the present invention is a DNA sequence optimized through Escherichia coli preferred codons, specifically as follows:

First of all, the wild-type HPV 58L1 gene was modified, and all its amino acids used the most frequently used nucleotide codons in Escherichia coli; at the same time, in order to avoid the high proportion of GC in the translated mRNA, the secondary structure of the mRNA It affects the translation efficiency, avoids some commonly used enzyme cutting sites, corrects the optimal codon frequency, and considers other influencing factors, and finally designs a new HPV58 L1 DNA sequence, such as SEQ ID Shown in NO.1.

The optimized HPV58L1 DNA was sent to Shenggong Bioengineering (Shanghai) Co., Ltd. for synthesis, and the synthesized gene was directly loaded into the pUC57 plasmid, which was named the recombinant plasmid pUC-58L1.

2. PCR amplification and double enzyme digestion recovery of HPV58 L1 gene

2.1 Design and synthesis of primer sequences

Design and synthesize forward primers according to the HPV58 L1 gene sequence, see Table 1 for details; wherein, the forward primer F-58 includes a Bsa I restriction endonuclease site; the reverse primer R-58 includes XhoI flanking the stop codon Restriction endonuclease site, the enzyme cutting site is shown underlined in the primer sequence.

Table 1 Primer list Primer name Restriction sites sequence (5'-3') F-58 <i>Bas I</i> TT<u><i>GGTCTC</i></u>TAGGTATGTCCGTGTGGCGTC (SEQ ID NO. 2) R-58 <i>Xho I</i> CCG<u><i>CTCGAG</i></u>TTATTTTTTAACCTTTTTGCGT (SEQ ID NO. 3)

2.2 PCR amplification and recovery of HPV58L1 gene

The pUC57-HPV58 L1 plasmid containing the HPV58L1 gene synthesized by the biological company is used as a template, and the F-58 and R-58 primers are used to amplify the target gene. The amplification system is as follows:

Component volume (50 μL)

Primer STAR Max 25 μL

F-58 1 μL

R-58 1 μL

pUC57-HPV58 L1 plasmid 1 μL (10ng)

Sterile DDW 22 μL.

The total reaction system was 50 μL, and the PCR reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 90 s; a total of 30 cycles; extension at 72°C for 10 min. After PCR, the obtained product was identified by electrophoresis. After identification, the successfully amplified L1 gene was recovered using a DNA recovery kit.

2.3 Recovery of PCR amplification product and double enzyme digestion of empty vector plasmid

The pESUMO plasmid and the PCR amplified product (HPV58 L1 gene) were digested with restriction endonucleases Bsa I and Xho I, respectively. The specific reaction conditions are as follows:

The enzymatic digestion system is:

HPV58L1 gene or pESUMO plasmid 20 μL

10×Buffer 5 µL

Bsa I 1 μL

Xho I 1 μL

Double distilled water 23 μL.

The total volume of the system is 50 μL, and the reaction conditions are 37° C. for 4 hours. Subsequently, the double digestion products of the empty vector plasmid pE-SUMO and the HPV58L1 gene were recovered, and ligated overnight at 16°C with T4 DNA ligase. The ligation reaction system (20 μL) was as follows:

HPV58L1 gene 3 μL

pE-SUMO 9 μL

10×T4 Buffer 2μL

T4 DNA Ligase 1 μL

Sterile DDW 5 μL.

The ligated product pE-SUMO-L1 was transformed into E.coli competent cell DH5α, and positive clones were selected by bacterial liquid PCR and double-enzyme digestion, and the plasmid was extracted and sent for positive sequencing. The recombinant plasmid pESUMO-58L1 was obtained when the sequencing was correct.

3. Construction of recombinant expression bacteria for HPV58 L1 protein

The recombinant plasmid pESUMO-58L1 was transformed into Escherichia coli competent cells BL21, spread on Amp ⁺ /LB solid medium and cultured, and a single colony was picked for bacterial liquid PCR identification and double enzyme digestion (see Figure 1), and the screening was positive For the colonies, the positive colony plasmids were extracted and sequenced. If the sequencing results were correct, they were named recombinant plasmid pSUMO-HPV58L1, which was a recombinant plasmid expressing soluble human papillomavirus 58 subtype L1 protein.

Example 2. Preliminary induced expression and identification of HPV58 subtype L1 protein

1. Expression and identification of HPV58L1 E. coli expression strain

The Escherichia coli expression strain pESUMO-58L1 was inoculated in Amp ⁺ /LB liquid medium and cultured until the OD ₄₅₀ value was about 0.8, and then IPTG was added to make the final concentration 0.3 mmol/L, and then the expression was induced at 18°C for 12 hours; After the expression, the bacteria were collected by centrifugation, and a small part of the bacteria was identified by SDS-PAGE and West-Blotting. At the same time, empty E. coli was set up as a control. The results are shown in Figure 2. It can be seen from the figure that pESUMO-58L1 The expression product of the expression product has a target band at about 80kDa, and the HPV58L1 protein is about 58kDa; the SUMO tag is about 12kDa, plus the HIS-tag, the fusion protein SUMO-58L1 protein has a molecular weight of about 72kDa, because the HIS-tag is positively charged, in SDS- In the PAGE electrophoresis, the swimming rate lagged behind, so the predicted position of the target band at 80kDa was basically the same, indicating that the recombinant protein of SUMO-58L1 was expressed in the pESUMO-58L1 expression strain.

2. Solubility analysis of recombinant protein HPV58-L1

The concentration of the inducer IPTG was 0.3 mmol/L, and the expression of pESUMO-58L1 was induced at 16°C for 18 hours. The expression cells of pESUMO-58L1 were collected, resuspended in PBS, and subjected to sonication, and the supernatant and precipitate were identified by 12% SDS-PAGE, respectively. The results showed that there were protein bands at about 80kDa in the sonicated supernatant and the precipitate of pESUMO-58L1, while the expression level of the recombinant SUMO-58L1 protein in the supernatant was higher than that in the precipitate as a whole (Figure 2C).

Example 3. Purification and Activity Identification of HPV58 L1 Recombinant Protein

1. Purification of HPV58 L1 Recombinant Protein

Mix the remaining cells with the washing buffer at a weight ratio of 1:5, shake well, centrifuge at 12000r/min for 5min, and collect the cell pellet. Mix the bacterial pellet and the bacteriostasis buffer at a weight ratio of 1:10, shake well, and crush the bacterium under high pressure. Centrifuge the high-pressure crushed bacteriostasis solution at 12,000 r/min and 4°C for 30 minutes, and collect the supernatant.

The supernatant was purified by a Ni affinity chromatography column, and the elution method was: Wash Buffer, pH 8.0 containing 250 mmol/L NaCl 50 mmol/L Tris-HCl buffer + 20 mmol/L imidazole; Elution Buffer pH8. 0 containing 250mmol/LNaCl 50mmol/L Tris-HCl buffer+200mmol/L imidazole) for elution, collected the eluted fractions, and used SDS-PAGE, Western-blot detection, the results are shown in Figure 3, The molecular weight of the purified SUMO-23 L1 protein was in line with the expected result at 80kDa, which proved that the SUMO-23 L1 protein was successfully obtained.

2. Excision of SUMO-tag of SUMO-58 L1 Recombinant Protein

After Ulp1 protease was purified by Ni-NTA, an enzyme digestion reaction system was constructed to digest SUMO-L1 protein to remove SUMO-tag. The enzyme digestion system is: the ratio of Ulp1 protease to SUMO-L1 protein is 1:50, the enzyme digestion system contains 50 mmol/L Tris-HCL, 200 mmol/L NaCl and 1 mmol/L DTT, and the pH condition is 8.0 , after the enzyme digestion system was reacted at 30°C for 3 h, the enzyme digestion reaction system was sampled for 12% SDS-PAGE analysis. After the recombinant SUMO-L1 protein was reacted with Ulp1 protease, the enzyme digestion reaction solution was passed through a Ni-NTA chromatography column to separate SUMO-tag, Ulp1 protease and L1 protein in the enzyme digestion system, and the collected L1 Proteins are concentrated through Amicon® Ultra centrifugal filters to increase protein concentration.

The results are shown in Figure 4. Ulp1 had a single band at 48 KDa after purification with a Ni column, and the purity was about 90% (Figure 4A); after Ulp1 digested the recombinant SUMO-L1 protein, SDS-PAGE results showed that Ulp1 could digest The recombinant HPV58 L1 protein with the SUMO tag was specifically cleaved. After digestion, the size of the HPV58 L1 target protein was about 58 kDa, and the size of the SUMO tag was about 12.4 kDa (Figure 4B). The components such as SUMO-tag and Ulp1 protease in the system were separated from L1 protein, and the collected L1 protein was concentrated by Amicon® Ultra centrifugal filter, and the protein purity was about 95%.

3. Hemagglutination test (Hemagglutination, HA) to identify recombinant HPV58 L1 protein activity

3.1 Red blood cell preparation method

Use a sterilized syringe to draw 1ml of fresh mouse blood into a sterilized centrifuge tube containing 1,000U of heparin (anticoagulant); add 9ml of PBS to the centrifuge tube and centrifuge at 1500r/min for 5min, discard the supernatant; use 10ml Resuspend the blood cells in PBS, centrifuge at 2000r/min for 10min, discard the supernatant, and wash the red blood cells three times; finally, according to the required amount, use PBS to make a mouse red blood cell suspension with a volume fraction of 1%.

3.2 HA

Hemagglutination test: Take a 96-well miniature hemagglutination reaction plate, use a micropipette to absorb 25 μl of PBS, and add it from the first well to the eighth well, 25 μl per well. Pipette 25 μl of purified HPV58 L1 recombinant protein into well 1, mix well, then aspirate 25 μl from well 1, add to well 2 and mix thoroughly again, aspirate 25 μl from well 2, add to well 3 and mix well, By analogy, doubly dilute to the fifth well, and add 25 μl of PBS to each well of the sixth, seventh, and eighth wells to set it as a negative control. 25 μl of 1% mouse erythrocyte suspension was poured from the 8th well to the 1st well and suspended, vortexed and mixed; stood at room temperature for 40 minutes, took pictures and recorded the experimental results. The hemagglutination test showed that the purified HPV58 L1 protein had the hemagglutination activity of agglutinating mouse red blood cells, and the hemagglutination value was 1:16 (Figure 5).

Embodiment four, the animal experiment of HPV58 L1 recombinant protein

1. Immunization procedure

Animal grouping: Divide the mice to be immunized (female Balb/c mice aged 6-8 weeks) into three groups, 4 in each group, and the immunization doses of the three groups are 20 μg/mouse, 5 μg/mouse and NC group respectively (PBS). Immunization method: In the experiment, the experimental mice were immunized by subcutaneous multi-point injection, a total of 3 times, with an interval of two weeks between each time. Serum sample collection: Tail docking of mice on d 0, 7 d, 14 d, 21 d, 28 d and 36 d respectively Blood collection: after tail docking of mice, pipette the tail end of mice 10 μL of blood was diluted in 990 μL of PBS buffer (1:100 dilution), and then the collected blood samples were centrifuged at 3000 r/min for 10 min, and the supernatant was drawn, which was the 1:100 diluted serum sample, The obtained serum samples were marked and stored in a -80°C ultra-low temperature freezer.

2. Initial immune evaluation

(1) Determination of serum titer by ELISA

Dilute the L1 protein with CBS solution to a concentration of 4 μg/mL, add 100 μL per well into a 96-well plate, and coat at 37°C for 2 hours. Wash 5 times with PBST, add 300 μL per well of 5% skimmed milk solution to block overnight at 4 °C. Mouse immune serum was serially diluted 2 times as the primary antibody and incubated at 37°C for 1 h at a dilution of 1:3200 to 1:204800, and PBS was used as a negative control (NC). Wash 5 times with PBST, add HRP-goat anti-mouse IgG (1:5000 dilution) at 100 μL per well, and incubate at 37°C for 1 h. Wash 5 times with PBST, add 50 μL of TMB substrate chromogenic solution to each well and react for 5 min, add 2mol/L H ₂ SO ₄ to 50 μL per well to terminate the reaction, and read the absorbance value at 450 nm. When the OD ₄₅₀ value of the detection well is ≥ 2.1 times the OD ₄₅₀ value of the negative NC well (P/N ≥ 2.1), it is judged as a positive well, and the antibody dilution factor corresponding to the well is the antibody titer.

The results of ELISA showed that compared with the PBS group, both the 20 μg group and the 5 μg group produced specific antibodies, and with the increase in the number of immunizations and the lapse of immunization time, the level of specific antibodies also increased (Fig. 6A); on the 36th day after immunization, the serum titer of the mice could reach 1:1.024×10 ⁵ ( FIG. 6B ).

(2) Indirect immunofluorescence assay (IFA)

293T cells were seeded in 96-well cell plates at 2×10 ⁴ cells/well. After growing to about 60% confluence, mix the plasmids pcDNA3.1-GFP and pcDNA3.1-HPV58 L1 prepared in advance with Lipofectamine 2000 respectively, add serum-free DMEM, mix well and let stand for 20 minutes. The supernatant mixture was added to the cell culture plate, and complete DMEM medium was added after 6 h. After 48 hours of transfection, the transfection was judged by observing the expression of GFP, and the transfection of pcDNA3.1 empty load was used as a negative control. The transfected cells were fixed with pre-cooled methanol for 20 min, blocked with 5% skimmed milk at 37°C for 1 h, and the primary antibody was the immune serum diluted 1:200 with PBS, and incubated at 37°C for 1 h. PBS was used to immunize mouse serum as a negative control (NC), PBS was used instead of mouse serum as a blank control (BC), the secondary antibody was diluted 1:100 with FITC-goat anti-mouse IgG, incubated at 37°C for 1 h, washed with PBS for 3 The cell morphology and fluorescence intensity were observed with an inverted fluorescence microscope.

The results of IFA showed that the GFP protein in the control group was successfully expressed, indicating that the transfection was successful (Fig. 7A); while 293T cells were transfected with pcDNA3.1-HPV58 L1 for transient expression and then incubated with mouse immune serum, an obvious green color could also be observed Fluorescence, indicating that eukaryotic HPV58 L1 can specifically react with mouse immune serum (Figure 7B), indicating that mice can produce specific antibodies in vivo after immunostimulation of HPV58 L1 protein obtained through prokaryotic expression.

The present invention has been described in detail above in conjunction with the accompanying drawings and embodiments. However, those skilled in the art can understand that without departing from the concept of the present invention, the specific parameters in the above embodiments can also be changed. Alternatively, relevant steps, methods, and materials are equivalently substituted to form multiple specific embodiments, all of which are common variation scopes of the present invention, and will not be described in detail here.

SEQUENCE LISTING

<110> Zhengzhou University

<120> Construction and application of gene encoding soluble HPV58 L1 protein and its recombinant plasmid

<130> /

<160> 3

<170> PatentIn version 3.2

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<211> 1497

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<213>HPV58L1

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cgtctgctgg ctgttggcaa tccatacttc tccatcaaat ctccgaacaa caacaaaaaa 180

gtactggttc cgaaggtatc tggcctgcag tatcgtgtct ttcgtgtgcg tctgccggat 240

cccaacaagt tcggtttccc ggacaccagc ttctacaacc cggataccca acgtctggtc 300

tgggcatgtg taggcctgga aatcggtcgt ggtcagccac tgggtgttgg cgtatctggt 360

catccgtatt tcaacaaatt tgatgacact gaaacctcta accgttatcc ggcacagcca 420

ggttctgata accgtgaatg cctgtctatg gattataaac aaacccaact gtgtctgatt 480

ggctgtaaac cgccgactgg tgagcattgg ggtaaaggtg ttgcctgtaa caacaacgca 540

gctgctactg attgtccgcc actggaactg tttaactcta ttattgagga tggtgacatg 600

gtagataccg gttttggttg catggacttt ggtaccctgc aggctaacaa atctgatgtg 660

ccgattgata tttgtaactc tacctgcaaa tatccagatt atctgaaaat ggcctctgaa 720

ccgtatggtg attctctgtt cttttttctg aggcgtgagc agatgttcgt tcgtcacttc 780

ttcaaccgtg ccggtaaact gggcgaggct gtcccggatg acctgtatat taaaggttcc 840

ggtaacactg cagttatcca atcttctgca ttttttccaa ctccgtctgg ctctatggtt 900

acctctgaat ctcaactgtt taacaagccg tattggctgc agcgtgcaca aggtcataac 960

aacggcattt gttggggcaa tcagctgttc gtgaccgtag ttgataccac tcgtagcact 1020

aacatgaccc tgtgcactga agtaactaag gaaggtacct ataaaaacga taactttaag 1080

gaatatgtac gtcatgttga agaatacgac ttacagttcg tgttccagct gtgcaagatt 1140

accctgactg cagagatcat gacctatatc catactatgg attccaacat tctggaggac 1200

tggcaatttg gtctgacccc gccgccgtct gcctctctgc aggacaccta tcgttttgtt 1260

acctcccagg ctattacttg ccaaaaaacc gcaccgccga aagaaaaaga agatccactg 1320

aacaaatata ctttttggga ggttaacctg aaggaaaagt tttctgcaga tctggatcag 1380

tttccgctgg gtcgtaagtt tctgctgcaa tctggcctga aagcaaagcc gcgtctgaaa 1440

cgttctgccc cgactacccg tgcaccatcc accaaacgca aaaaggttaa aaaataa 1497

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ccgctcgagttattttttaa cctttttgcg t 31

Claims (8)

1. The gene HPV58L1 for coding soluble human papilloma virus 58 subtype L1 protein has the DNA sequence shown in SEQ ID NO. 1.

2. A vector plasmid pUC-58L1 comprising vector plasmid pUC57 and the gene HPV58L1 of claim 1 loaded therein.

3. A recombinant plasmid pESUMO-58L1, comprising a vector plasmid pE-SUMO and the HPV58L1 gene of claim 1 loaded therein.

4. A recombinant plasmid for expressing soluble HPV58L1 protein, which contains the gene HPV58L1 of claim 1.

5. The method for constructing the recombinant plasmid expressing the soluble HPV58L1 protein according to claim 4, comprising the following steps:

(1) Designing an amplification primer based on the gene HPV58L1 of claim 1, and a forward primer thereof comprisesBsa IRestriction enzyme site, reverse primer including flanking stop codonXho IA restriction enzyme site;

(2) PCR amplifying gene HPV58L1 by using the amplification primer;

(3) Respectively carrying out restriction endonuclease on pESUMO plasmid and obtained PCR amplification productBsa IAnd withXhoI double digestion, recovery of empty vector plasmid pE-SUMO and HPV58L1 gene doubleEnzyme digestion products are connected overnight at 16 ℃ by T4 DNA ligase to obtain recombinant plasmids;

(4) The recombinant plasmid pESUMO-58L1 obtained in the previous step is transformed into escherichia coli competent cells BL21 and coated on Amp ⁺ Culturing on LB solid culture medium, selecting single colony to carry out bacteria liquid PCR identification, screening positive colony, extracting positive colony plasmid, and obtaining recombinant plasmid expressing soluble HPV58L1 protein with correct sequencing result.

6. The method for constructing a recombinant plasmid according to claim 5, wherein the sequences of the amplification primers are as follows:

F-58：5’- TT GGTCTC TAGGTATGTCCGTGTGGCGTC-3', enzyme cutting site:Bas I；

R-58：5’- CCG CTCGAG TTATTTTTTAACCTTTTTTTTGCGT-3', cleavage site:Xho I。

7. a preparation method of soluble HPV58L1 protein comprises the following steps:

(1) Inoculating the recombinant plasmid pESUMO-58L1 of claim 3 to Amp ⁺ Culturing in LB liquid culture medium to OD ₄₅₀ When the value reaches 0.75-0.85, inducing expression by an inducer IPTG;

(2) After induction expression is finished, centrifugally collecting thalli, washing, crushing, centrifugally separating and collecting supernate;

(3) And eluting and purifying the supernatant through a Ni affinity chromatography column to obtain the SUMO-58L1 protein.

8. The use of the gene HPV58L1 as claimed in claim 1, the vector plasmid pUC-58L1 as claimed in claim 2, the recombinant plasmid pESUMO-58L1 as claimed in claim 3 or the soluble HPV58L1 protein as claimed in claim 7 in the preparation of vaccine.

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