CN110156879A - Preparation method and application of the C-terminal fragment of Kaposi's sarcoma-associated virus protein ORF57 and its antibody - Google Patents

Abstract

The invention discloses a preparation method and application of an antibody of a C-terminal fragment of Kaposi's sarcoma-associated virus protein ORF57. The polypeptide fragment is obtained by cloning the gene of the polypeptide fragment and realizing prokaryotic expression and purification. Using the polypeptide fragment as an antigen, prepare rabbit antiserum, separate and purify the antiserum, and obtain polyclonal antibody against ORF57 protein. Experimental studies of cytology and molecular biology show that the antibody can specifically bind ORF57 protein inside and outside the cell, and can detect ORF57 protein entering the nucleus. Therefore, the polyclonal antibody can be used in the detection and recognition of Kaposi's sarcoma-associated virus antigen or in the preparation of a detection kit for detection of live virus infection.

Description

Kaposi's sarcoma-associated virus protein ORF57 C-terminal fragment and antibody preparation method thereof application

technical field

The invention relates to the field of biomedicine, in particular to a polyclonal antibody preparation method and application of the C-terminal fragment of the ORF57 protein of Kaposi's sarcoma-associated virus.

Background technique

Kaposi's sarcoma (Kaposi's sarcoma, KS) is a common complication of AIDS (Acquired Immune Deficiency Syndrome, AIDS) patients. Kaposi’s sarcoma-associated herpesvirus (KSHV); also known as Human herpesvirus 8 (HHV8) is the causative agent of this tumor. In addition, KS is one of the most common cancers in many subequatorial African countries. Human immunodeficiency virus (human immunodeficiency virus, HIV) infection is considered to be one of the most important factors that can significantly increase the risk of KS. The incidence of KS is 1/100,000 in the general population, rising to 1/20 in HIV-infected men, and climbing to 1/3 in HIV-infected gay men. Men co-infected with HIV and KSHV have a 50% chance of developing KS within ten years if they do not receive effective treatment.

KS is mainly composed of spindle cells, abnormally proliferating blood vessels, and inflammatory cell infiltration. KSHV has a two-phase life cycle: a latent phase and a lytic phase. KSHV can regulate the biphasic life cycle according to the environment. When the viral lytic phase occurs, the open reading frame 57 protein (open reading frame 57, ORF57) is expressed early in the viral lytic phase. Protein ORF57 can improve the stability of viral mRNA to promote KSHV to complete cleavage and replication, and viruses lacking ORF57 gene cannot effectively produce mature virions. The study also found that ORF57 regulates the expression of interleukin 6 and various host proteins, and inhibits host cell antiviral defense. Therefore ORF57 protein plays an important role in the infection of KSHV.

At present, there are very few research results on the function and biological characteristics of the protein ORF57. Further in-depth research requires specific antibodies against this protein, which are not yet commercially available.

Contents of the invention

One of the objectives of the present invention is to provide a C-terminal fragment targeting Kaposi's sarcoma-associated herpes virus protein ORF57.

The second object of the present invention is to provide the encoded protein of the C-terminal fragment of the herpes virus protein.

The third object of the present invention is to provide a vector comprising the C-terminal fragment.

The fourth object of the present invention is to provide a method for cloning the C-terminal fragment.

In the present invention, an antigenic peptide is firstly designed for the C-terminal domain of the protein ORF57, and the gene is molecularly cloned to realize high-efficiency prokaryotic expression. A highly pure C-terminal fragment of the recombinant protein ORF57 can be obtained.

To achieve the above object, the present invention adopts the following technical solutions:

A C-terminal fragment of Kaposi's sarcoma-associated virus protein ORF57, characterized in that the gene of the fragment is the base sequence shown in SEQ ID NO:1.

The above-mentioned encoded protein of the gene of the C-terminal fragment of Kaposi's sarcoma-associated virus protein ORF57 is characterized in that the encoded protein is the amino acid sequence shown in SEQ ID NO:2.

A carrier, characterized in that the carrier contains the above-mentioned base sequence.

A recombinant plasmid, characterized in that the recombinant plasmid contains the above-mentioned vector.

A method for cloning the C-terminal fragment of the above-mentioned Kaposi's sarcoma-associated virus protein ORF57, characterized in that the specific steps of the method are:

a. design specific primers containing restriction endonuclease sites, the specific primers are the base sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4:

b. Perform PCR amplification using the specific primers in step a to obtain and purify the C-terminal antigen fragment of the ORF57 gene.

In the above step b, 1.0 mM IPTG was used for PCR amplification, the induction temperature was 30° C., and expression was performed for 4 hours.

In the above-mentioned step b, a Ni column is used for purification.

An application of the above-mentioned C-terminal fragment of Kaposi's sarcoma-associated virus protein ORF57 in detecting and identifying live virus infection.

Preferably, the nucleotide sequence of the gene of the C-terminal fragment of the protein ORF57 is as shown in SEQ ID NO:2. The gene of the C-terminal fragment of the protein ORF57 is to intercept the C-terminal fragment part of the whole gene of the protein ORF57, and it is cloned by polymerase chain reaction, and the two primers used to clone the gene fragment respectively contain BamHI and XhoI enzyme cutting sites point, the sequence distribution is shown in SEQ ID NO:3 and SEQ ID NO:4.

Preferably, the prokaryotic expression is induced by IPTG, the final concentration of IPTG is 1 mM, and the culture temperature for inducing expression is 30°C.

The present invention provides a purification method of the above-mentioned soluble recombinant defense, the purification method comprising the following steps:

The Escherichia coli BL21 expressing the soluble recombinant protein was collected by centrifugation, the collected thalline was bathed in ice, subjected to ultrasonic lysis, and the supernatant was separated and collected. The supernatant was purified on a Ni column, and the above-mentioned soluble recombinant protein could be obtained by eluting with an eluent, and the effective eluent contained 0.3M imidazole.

The present invention adopts the above-mentioned prokaryotic expression protein system to prepare the C-terminal fragment antigen of protein ORF57.

The present invention provides an application of the above-mentioned C-terminal fragment antigen of protein ORF57 in the preparation of polyclonal antibody against ORF57.

The present invention provides a method for preparing a polyclonal antibody specific for the C-terminal fragment of recombinant protein ORF57, the method comprising the following steps:

After emulsifying the C-terminal fragment of recombinant protein ORF57 as a protein antigen with Freund's adjuvant (Freund's adjuvant), it was injected subcutaneously at multiple points on the back of New Zealand white rabbits. After the initial immunization, three booster immunizations were carried out. After the antigen immunization process, rabbit blood was collected from the carotid artery, coagulated, and centrifuged to obtain antiserum containing the C-terminal fragment of the specific rabbit anti-protein ORF57, which was affinity-purified by protein A affinity chromatography to obtain polyclonal antibodies .

The application of the polyclonal antibody prepared by the above method in the detection and recognition of KSHV virus antigen or the application in the preparation of ELISA detection kit for detection of KSHV live virus infection.

The polyclonal antibody identification prepared by the above method can be used for detection and recognition of KSHV virus antigen in Western Blot analysis and immunofluorescence analysis.

The present invention optimizes the C-terminal fragment gene of ORF57 of KSHV virus for the first time, expresses the protein by using the prokaryotic Escherichia coli expression system, and obtains the recombinant protein antigen retaining the specific three-dimensional structure. The polyclonal antibody prepared by immunizing New Zealand white rabbits with this antigen can be used for Western Blot detection and specific recognition of protein ORF57 in cells. Therefore, the C-terminal fragment antigen peptide of ORF57 and its polyclonal antibody can lay the foundation for the further development of KSHV detection kits.

Description of drawings

Fig. 1 is the electrophoresis detection result of the PCR identification of the constructed recombinant plasmid pET28a-ORF57-C. Lane 1 is the DNA marker, and lane 2 is the PCR product of the recombinant vector.

Fig. 2 shows the antigenic peptide of the C-terminal domain of the specifically expressed recombinant protein ORF57 by SDS-PAGE electrophoresis. Among them, the M lane is the protein marker, the B lane is the agarose substrate after hanging the column, the E lane is the eluent (0.3M imidazole), the W lane is the eluent (0.12M imidazole), the O lane is the flow-through, and the P Lane is the inclusion body, lane S is the supernatant, lane T is the sample after ultrasonic lysis, and lane C is the lysate of the control group.

Figure 3 is the titer determination of specific polyclonal antibodies.

Figure 4 is the detection of the specificity of polyclonal antibodies by Western blot. Lane M in the figure is the protein marker, lane 1 is the control group, and lane 2 is the experimental group expressing ORF57 protein.

Fig. 5 Fluorescence immunoassay shows that the polyclonal antibody can specifically recognize the protein ORF57 localized in the nucleus. In the figure: A is the cell nucleus stained with DAPI, B is the ORF57 recombinant protein with green fluorescence in the nucleus, C is the cell nucleus labeled with rabbit polyclonal antibody and red fluorescent secondary antibody, D is B and C Effect diagram after stacking.

Specific implementation method

Below in conjunction with specific embodiment, further set forth the present invention. It should be understood that these examples are only used to explain the present invention, not to limit the scope of the present invention. Under the premise of not departing from the technical solution of the present invention, any modification made to the present invention that can be easily realized by those skilled in the art will fall within the scope of the claims of the present invention.

Embodiment one: the construction of the expression vector of the C-terminal structural domain antigen peptide of protein ORF57

The gene fragment sequence of the C-terminal domain antigen peptide is amplified from the full-length ORF57 gene of KSHV by PCR technology, and the nucleotide sequence is shown in SEQ ID NO:1. DNA restriction endonucleases BamHI and XhoI restriction sites were respectively introduced when designing PCR amplification primers, and the nucleotide sequence was as shown in SEQ ID NO:3. The gene fragment obtained after amplification was named ORF57-C.

The amplified PCR product was double-digested with restriction endonucleases BamHI and XhoI, then recovered and purified, and treated with the same double-digestion to achieve the expression plasmid pET28a, ligated overnight at 4°C, and the ligated product was transformed into competent E. Coli. DH5α bacteria were spread on LB plates containing kanamycin sulfate and cultured overnight at 37°C. Pick a single colony on the plate, culture it in liquid LB medium containing kanamycin sulfate for about 4 hours, and then carry out PCR identification and sequencing of the bacteria liquid. The bacteria solution with correct sequencing was added to fresh LB medium containing kanamycin sulfate at a ratio of 1:1000, cultured with shaking overnight, and then the bacteria were collected to extract the plasmid and save it for later use. The recombinant plasmid was named pET28a-ORF57-C. The harvested plasmids were amplified by PCR, and the amplified products were identified by agarose gel electrophoresis. The identification results are shown in Figure 1. It can be seen from the figure that the only characteristic band can be seen at about 800 bp, which is in line with expectations, indicating that ORF57-C was successfully cloned into the pET28a plasmid.

Example 2: Prokaryotic expression and purification of the recombinant protein of the C-terminal domain antigenic peptide of protein ORF57

The obtained recombinant plasmid was transformed into competent Escherichia coli BL21(E3), spread on LB plates containing kanamycin sulfate, and cultured at 37°C for 18h. Pick a single colony on the plate, culture it overnight in the liquid LB medium containing kanamycin sulfate, and re-inoculate it into the liquid LB medium containing kanamycin sulfate at 1:100 the next day, add 1.0mM IPTG, The temperature of the constant temperature shaker was 30°C, and the expression was performed for 4h. Collect the cells, wash and resuspend in an ice bath, and ultrasonically lyse the cells. The lysate was centrifuged several times and the supernatant was collected. Ni column of GE Company was used for purification, and 0.3M imidazole eluent was used for elution. The purified protein was detected by 10% SDS-PAGE electrophoresis. The gel was stained with Coomassie Brilliant Blue, and a dark band of the recombinant protein was visible at about 30KDa, which was basically invisible in control group C, as shown in Figure 2. Comparing different concentrations of imidazole elution solution, 0.3M imidazole elution solution can elute the characteristic protein.

Example 3: Preparation of Polyclonal Antibody to Antigen Peptide of C-terminal Domain of Protein ORF57

The purified C-terminal antigenic peptide of recombinant ORF57 was mixed with Freund's adjuvant to emulsify, and three New Zealand white rabbits of about 1.5Kg were used to prepare polyclonal antibodies, which were injected subcutaneously at multiple points on the back, and immunized three times in total. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second and third booster immunizations. Rabbit blood was collected by carotid artery bleeding, left at room temperature for 2 hours, overnight at 4°C, centrifuged to collect the supernatant serum, 56°C for 30 minutes to inactivate complement, and protein A column was used for affinity chromatography to collect protein peaks, aliquoted and stored in -80°C refrigerator.

Antibody titer was determined by ELISA method. Coat the ELISA plate with recombinant protein ORF57, block with 10% skimmed milk for 4 hours, add purified polyclonal antibodies of different dilutions, act at 37°C for 2 hours, wash 3 times, incubate with HRP-goat anti-rabbit IgG for 1 hour, OPD Color development, microplate reader detects the light absorption value, and calculates the titer of the antiserum. The result shows that the titer of the antibody is very high, as shown in Figure 3.

Embodiment four: Western blot detects the specificity of polyclonal antibody

Electrophoresis with HEK293T cell lysate induced to express protein ORF57, separated by 10% SDS-PAGE, semi-dry transfer to PVDF membrane, blocked overnight with 10% skimmed milk, immersed in diluted anti-polyclonal antibody, 25°C After acting for 2 hours and fully washing, add fluorescent secondary antibody and use Odyssey infrared laser detection system for detection. The results are shown in 4. The figure shows that a specific band can be detected in the induced cell lysate, the molecular weight is about 30kD, and there is no band at the same molecular weight in the negative control group. This result shows that the prepared polyclonal antibody can The ORF57 protein in the cells was specifically detected, as shown in Figure 4.

Embodiment five: immunofluorescence test detects the specificity of polyclonal antibody

HEK293T cells induced to express the protein ORF57 were collected, and the cells were seeded in a 24-well cell plate pre-placed in glass cell slides, and the cell culture plate was placed at 37°C and cultured in 5% CO ₂ for 24 hours. After the cell slides were washed with phosphate buffer, 4% paraformaldehyde was added dropwise, fixed at room temperature for 1 hour, and fully washed. Add 0.2% Trition X-100 to act for 30min, wash with PBS, add dropwise the prepared polyclonal antibody at 1:300 dilution, incubate at 37°C for 2h, after washing thoroughly, add goat anti-rabbit IgG-Alex Fluor at 1:1000 dilution , incubate in a 37°C incubator for 1 h, add 0.2 mg/ml DAPI dropwise to stain the nuclei in the dark for 5 minutes, add anti-fade agent dropwise after washing, and observe with a laser confocal microscope. It can be seen in the figure that the KSHV protein ORF57 characteristically enters the nucleus, and the prepared polyclonal antibody can specifically recognize the ORF57 protein located in the nucleus, the results are shown in Figure 5.

sequence listing

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Claims (8)

1. a kind of C-terminal segment of Kaposi's sarcoma associated viral protein ORF57, it is characterised in that the gene of the segment is SEQ Base sequence shown in ID NO:1.

2. the coding egg of the gene of the C-terminal segment of Kaposi's sarcoma associated viral protein ORF57 according to claim 1 It is white, it is characterised in that the coding albumen is amino acid sequence shown in SEQ ID NO:2.

3. a kind of carrier, it is characterised in that the carrier contains base sequence according to claim 1.

4. a kind of recombinant plasmid, it is characterised in that the recombinant plasmid includes carrier according to claim 3.

5. a kind of cloning process of the C-terminal segment of Kaposi's sarcoma associated viral protein ORF57 according to claim 1, It is characterized in that the specific steps of this method are as follows:

A. the specific primer containing restriction endonuclease sites is designed, which is SEQ ID NO:3 and SEQ ID Base sequence shown in NO:4:

B. PCR amplification is carried out using the specific primer of step a, obtains the C-terminal antigen fragment of ORF57 gene and purifies.

6. according to the method described in claim 5, it is characterized in that in the step b PCR amplification use 1.0mM IPTG, Inducing temperature is 30 DEG C, expresses 4h.

7. according to the method described in claim 5, it is characterized in that using Ni column purification in the step b.

8. a kind of C-terminal segment of Kaposi's sarcoma associated viral protein ORF57 according to claim 1 is detecting and is knowing Application in other live virus infection.