H5N1 nucleoprotein prokaryotic expression vector and method for inducing expression of nucleoprotein by using the same

Abstract

The present application relates to a kind of H5N1 nucleoprotein prokaryotic expression vector and the method for its nucleoprotein induced expression, belong to the field of bioengineering, the H5N1 nucleoprotein base sequence optimized by in vitro synthesis in the present application, by enzyme cutting site BamHI and XhoI, the H5N1 nucleoprotein optimized base sequence is constructed into PGEX-6P-1 plasmid and obtains H5N1 nucleoprotein prokaryotic expression vector;After being transformed into BL21 competent bacteria by H5N1 nucleoprotein prokaryotic expression vector, inoculate in LB solid culture medium with ampicillin resistance and culture, pick single bacterial clone and inoculate into LB liquid culture medium with ampicillin resistance and culture, finally, IPTG culture induction is obtained, and the nucleoprotein fusion protein of induced expression is obtained, and the nucleoprotein of H5N1 is well expressed, and the fusion protein is obtained, which provides the basis for studying more significant, more durable immunity and effective H5N1 vaccine.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically, it relates to an H5N1 nuclear protein prokaryotic expression vector and a method for inducing nuclear protein expression. Background Technology

[0002] Over the past century, highly pathogenic avian influenza (H5N1) has caused various outbreaks in eight countries, caused by different H5 viruses. The first outbreak of avian influenza was in 1959, caused by the H5N1 strain in Scottish chickens. Furthermore, since 2021, the H5N1 subtype of H5N1 H5N1 has caused a whole-animal epidemic in birds, disrupting poultry production worldwide and infecting multiple mammalian host species. An outbreak in U.S. dairy cows infected thousands in 16 states and subsequently spread to birds and other mammals. Human H5N1 infections have increased with increasing frequency of contact with infected animals. The risk of an H5N1 pandemic continues to rise. Since 1997, there have been approximately 900 human cases of H5N1 globally, with a case fatality rate exceeding 50%, demonstrating the dual threat of H5N1 to both animal and human health. While sustained human-to-human transmission has not been observed, the severity of this disease underscores the need for continued vigilance and robust public health measures.

[0003] Vaccines are the primary defense against influenza, and most infections can be prevented through vaccination. The most advanced technology for preparing influenza vaccines involves using recombinant DNA technology to produce immunogenic peptide components of the virus in microorganisms. In recent years, there has been significant interest and attention regarding the utilization of conserved epitope sequences of influenza virus antigenic proteins. Matrix protein 2 (M2e) sequences and peptide components derived from influenza virus nucleoprotein (NP) and neuraminidase (NA) proteins can be used to prepare recombinant peptide vaccines to induce more significant and longer-lasting immunity and efficacy. NPs are viral nucleoproteins that, together with viral RNA polymerase, form a ribonucleoprotein complex. Ubiquitination and deubiquitination of the NP protein regulate influenza A virus RNA replication.

[0004] Therefore, obtaining a prokaryotic expression vector for H5N1 nucleoprotein is of positive significance for researching H5N1 avian influenza vaccines with more significant, longer-lasting immunity and efficacy. Summary of the Invention

[0005] To address the problems existing in the background art, the present invention provides an H5N1 nucleoprotein prokaryotic expression vector and a method for inducing nucleoprotein expression, which provides an important foundation for the research of H5N1 avian influenza vaccines with more significant, longer-lasting immunity and efficacy.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] The H5N1 nuclear protein prokaryotic expression vector was obtained by constructing the optimized base sequence of the H5N1 nuclear protein into the PGEX-6P-1 plasmid through the restriction enzyme sites BamHI and XhoI; the optimized base sequence of the H5N1 nuclear protein is shown in SEQ ID NO. 1.

[0008] Furthermore, the optimized base sequence of the H5N1 nucleoprotein was obtained through in vitro synthesis.

[0009] A method for constructing a prokaryotic expression vector for H5N1 nucleoprotein includes the following steps:

[0010] (1) The optimized base sequence of H5N1 nucleoprotein was synthesized in vitro;

[0011] (2) The optimized base sequence of H5N1 nucleoprotein was constructed into the PGEX-6P-1 plasmid by using the restriction sites BamHI and XhoI.

[0012] The method for inducing nuclear protein expression using the above-mentioned H5N1 nuclear protein prokaryotic expression vector involves transforming the H5N1 nuclear protein prokaryotic expression vector into BL21 competent bacteria, inoculating them into LB solid medium with ampicillin resistance, selecting single bacterial clones and inoculating them into LB liquid medium with ampicillin resistance, and finally adding IPTG to induce the expression of the induced nuclear protein fusion protein.

[0013] Furthermore, the method for inducing nuclear protein expression includes the following steps:

[0014] (1) The H5N1 nuclear protein prokaryotic expression vector was added to BL21 competent bacteria and incubated on ice;

[0015] (2) Heat shock at 40-44℃;

[0016] (3) Incubation on an ice bath;

[0017] (4) Incubate in a shaker at 35-39℃;

[0018] (5) Spread the competent bacteria obtained in step (4) evenly on LB solid culture dishes with ampicillin resistance, and incubate overnight in an inverted incubator at 35-39℃;

[0019] (6) Select a single bacterial clone and inoculate it into LB liquid medium with ampicillin resistance and incubate it in a shaker at 35-39℃;

[0020] (7) After inoculating the culture medium from step (6) into LB liquid medium with ampicillin resistance and culturing it in a shaker at 35-39℃, add IPTG to induce it;

[0021] (8) Incubate in a shaker at 23-27℃ to obtain the induced expression of the nucleoprotein fusion protein.

[0022] The present invention also protects fusion proteins obtained by the above methods.

[0023] The application of the fusion protein described above in the preparation of drugs for the prevention or treatment of H5N1.

[0024] The present invention also provides an H5N1 vaccine comprising the fusion protein described above.

[0025] The beneficial effects of this invention are:

[0026] This invention synthesizes an optimized base sequence of H5N1 nucleoprotein in vitro, and constructs the optimized H5N1 nucleoprotein base sequence into the PGEX-6P-1 plasmid through the restriction enzyme sites BamHI and XhoI to obtain a prokaryotic expression vector of H5N1 nucleoprotein. The expression vector is successfully expressed in the PGEX-6P-1 plasmid.

[0027] This invention involves transforming the H5N1 nucleoprotein prokaryotic expression vector into BL21 competent bacteria, inoculating them into LB solid medium with ampicillin resistance, culturing single bacterial clones into LB liquid medium with ampicillin resistance, and finally adding IPTG to induce the expression of the nucleoprotein fusion protein, thus achieving good expression of the H5N1 nucleoprotein. Attached Figure Description

[0028] Figure 1 The results of double-enzyme digestion agarose gel electrophoresis of the H5N1 nuclear protein prokaryotic expression vector of the present invention are shown.

[0029] Figure 2 These are the SDS-PAGE results of the H5N1 nuclear protein induced by the present invention;

[0030] Figure 3 This is the WB result (nuclear protein antibody) after H5N1 nucleoprotein induction expression according to the present invention.

[0031] Figure 4 This is the WB result (GST antibody) after the H5N1 nuclear protein was induced to express according to the present invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0033] To illustrate the present invention more clearly, the following embodiments will be described in detail.

[0034] Example 1: Construction of H5N1 nuclear protein prokaryotic expression vector

[0035] The nucleoprotein sequence of H5N1 was optimized according to the species characteristics of Escherichia coli, and the optimized base sequence is shown in SEQ ID NO.1.

[0036] Optimized base sequence of H5N1 nucleoprotein (SEQ ID NO.1):

[0037]

[0038] The optimized base sequence of H5N1 nucleoprotein was constructed into the PGEX-6P-1 plasmid by using the restriction sites BamHI and XhoI to obtain the PGEX-6P-1 plasmid that can express H5N1 nucleoprotein, namely the H5N1 nucleoprotein prokaryotic expression vector (PGEX-6P-1-H5N1-NP plasmid).

[0039] Identification was performed using double enzyme digestion, and the results are attached. Figure 1 This indicates that the H5N1 nucleoprotein base sequence has been successfully constructed into the PGEX-6P-1 plasmid, and the PGEX-6P-1 plasmid that can express the H5N1 nucleoprotein has been obtained, namely the PGEX-6P-1-H5N1-NP plasmid (H5N1 nucleoprotein prokaryotic expression vector).

[0040] Example 2: Induced expression of H5N1 nuclear protein

[0041] The PGEX-6P-1-H5N1-NP plasmid (H5N1 nuclear protein prokaryotic expression vector) was transformed into BL21 competent bacteria. The specific operation steps are as follows:

[0042] BL21 competent bacteria inoculated with the PGEX-6P-1-H5N1-NP plasmid (H5N1 nucleoprotein prokaryotic expression vector) were incubated on ice for 30 min, then heat-shocked at 42°C for 90 s, incubated on ice for 2 min, and then incubated on a shaker at 37°C for 60 min. The competent bacteria were then evenly spread onto LB solid culture dishes containing ampicillin and incubated upside down at 37°C overnight. A single bacterial clone was picked from the overnight culture dish and inoculated into 5 ml of LB liquid medium containing ampicillin and incubated on a shaker at 37°C for approximately 8 h. Immediately afterwards, 5 ml of the culture was inoculated into 50 ml of LB liquid medium containing ampicillin and incubated on a shaker at 37°C for approximately 2 h. IPTG was then added to a final concentration of 0.5 mM and incubated on a shaker at 25°C for approximately 4 h to induce N antigen expression, thus obtaining the induced nucleoprotein fusion protein. The induced fusion protein was detected by SDS-PAGE and WB, and the results are attached. Figure 2 As shown, the nucleoprotein was well expressed under the induction conditions of 0.5 mM IPTG and 25 °C. Simultaneously, we used Western blotting to detect the fusion protein and performed hybridization staining with H5N1 nucleoprotein antibody and GST protein antibody, respectively. The results are shown below. Figure 3 and Figure 4 As shown, the results indicate that the nucleoprotein of H5N1 was well expressed, and the acquisition of this fusion protein provides a foundation for studying H5N1 vaccines with more significant, longer-lasting immunity and efficacy.

[0043] To further evaluate the immunogenicity of the expressed H5N1 nuclear protein at the animal level, 7-week-old specific pathogen-free (SPF) BALB / c mice were selected and divided into two groups: an immunization group and a control group, with 5 mice in each group.

[0044] H5N1 nucleoprotein was formulated into samples containing 5 μg H5N1 nucleoprotein and 41 μg Al(OH)3 adjuvant per dose. Mice were immunized twice via intramuscular injection, with a 21-day interval between immunizations. The control group received no treatment. Serum samples were collected 14 and 28 days after the second immunization, and the binding antibody levels were detected using ELISA. The results showed that the level of binding antibodies against H5N1 nucleoprotein produced in mice 14 days after the second immunization was 1:819200, and the level was 1:713155 28 days after the second immunization. This indicates that the obtained H5N1 nucleoprotein has excellent immunogenicity, and the binding antibody level can be maintained at a high level in mice.

[0045] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A prokaryotic expression vector for H5N1 nuclear protein, characterized in that, The optimized base sequence of the H5N1 nucleoprotein was constructed into the PGEX-6P-1 plasmid by using the restriction enzyme sites BamHI and XhoI; the optimized base sequence of the H5N1 nucleoprotein is shown in SEQ ID NO.

1.

2. The H5N1 nuclear protein prokaryotic expression vector according to claim 1, characterized in that, The optimized base sequence of the H5N1 nucleoprotein was obtained through in vitro synthesis.

3. The method for constructing the H5N1 nuclear protein prokaryotic expression vector as described in claim 1 or 2, characterized in that, Includes the following steps: (1) The optimized base sequence of H5N1 nucleoprotein was synthesized in vitro; (2) The optimized base sequence of H5N1 nucleoprotein was constructed into the PGEX-6P-1 plasmid by using the restriction sites BamHI and XhoI.

4. A method for inducing nuclear protein expression in bacteria after transformation with the H5N1 nuclear protein prokaryotic expression vector as described in claim 1, characterized in that, The H5N1 nucleoprotein prokaryotic expression vector was transformed into BL21 competent bacteria and then inoculated into LB solid medium with ampicillin resistance. Single bacterial clones were picked and inoculated into LB liquid medium with ampicillin resistance. Finally, IPTG was added to induce the expression of the nucleoprotein fusion protein.

5. The method according to claim 4, characterized in that, Includes the following steps: (1) The H5N1 nuclear protein prokaryotic expression vector was added to BL21 competent bacteria and incubated on ice; (2) Heat shock at 40-44℃; (3) Incubation on an ice bath; (4) Incubate in a shaker at 35-39℃; (5) Spread the competent bacteria obtained in step (4) evenly on LB solid culture dishes with ampicillin resistance, and incubate overnight in an inverted incubator at 35-39℃; (6) Select a single bacterial clone and inoculate it into LB liquid medium with ampicillin resistance and incubate it in a shaker at 35-39℃; (7) After inoculating the culture medium from step (6) into LB liquid medium with ampicillin resistance and culturing it in a shaker at 35-39℃, add IPTG to induce it; (8) Incubate in a shaker at 23-27℃ to obtain the induced expression of the nucleoprotein fusion protein.

6. The method according to claim 5, characterized in that, The IPTG concentration in step (7) is 0.5 mM, and the induction temperature is 23-27℃.

Images