Application of LMH cell line overexpressing RPL7 gene and vaccine

Abstract

The invention belongs to the technical field of biology, and discloses application of an LMH cell line of an overexpressed RPL7 gene and a vaccine. Goose astrovirus is infected to an LMH cell line capable of overexpressing goose source RPL7, proliferation of the goose astrovirus can be promoted to a certain extent, RPL7 serves as a constituent part of a ribosome large subunit and directly participates in the synthesis process from mRNA to protein, GAstV mRNA and RPL7 interact and are located in an endoplasmic reticulum, the reverse transcription level of the virus is promoted to a large extent, and the effect of overexpressing the goose source RPL7 is achieved. Further, the replication of the goose astrovirus is promoted; when the goose astrovirus is applied to vaccine research and development, goose astrovirus with relatively high titer can be obtained, so that the cost of vaccine research and development can be reduced, and the effect of the vaccine can be improved.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, specifically to the use of LMH cell lines overexpressing RPL7 and vaccines. Background Art

[0002] Novel goose astrovirus (GAstV) belongs to the family Astroviridae, genus Avastrovirus, and is a member of avian astrovirus type 3; GAstV is a single-stranded positive-strand, non-enveloped RNA virus; currently, the lethal gosling gout disease caused by GAstV is still widespread, and the progress of vaccine research and development for this disease is particularly slow, and there is no available vaccine, and the prevention and control situation is very serious. At present, the only passable cell line available for GAstV proliferation is LMH cells, but the virus titer cultured by them is relatively low, which to a certain extent limits the efficient and in-depth research on the pathogenic mechanism of this virus, and also limits the efficiency and convenience of vaccine research and development for this disease. Therefore, increasing the proliferation titer of GAstV in LMH cells is of great significance for vaccine research and development, production, and research on the pathogenic mechanism of this disease.

[0003] Chinese Patent CN116970574B discloses the use of goose vimentin, a method and use for promoting the proliferation of goose astrovirus, and a vaccine. This scheme infects a cell line capable of expressing goose vimentin with goose astrovirus. Due to the direct interaction between vimentin and the viral capsid protein, it can promote the expression of the viral capsid protein of goose astrovirus, and thus promote the replication and proliferation of goose astrovirus.

[0004] Chinese Patent CN118126964B discloses the use of a cell line overexpressing RBM4, a method and use for promoting the proliferation of goose astrovirus, and a vaccine. This scheme infects goose astrovirus into an LMH cell line capable of overexpressing goose RBM4, thereby promoting the proliferation of goose astrovirus.

[0005] In summary, it can be seen that the research reports on using RPL7 to promote the proliferation of goose astrovirus are very limited. Therefore, the technical problem to be solved by the present invention is: how to develop a new method to promote the proliferation of goose astrovirus. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of an LMH cell line overexpressing goose RPL7 in the proliferation of goose astrovirus and a method for promoting the proliferation of goose astrovirus, so as to facilitate the cultivation of goose astrovirus with a relatively high titer through in vitro cell culture for the development of inactivated vaccines.

[0007] At the same time, the present invention also provides an LMH cell line and a preparation method thereof.

[0008] To achieve the above purpose, the present invention provides the following technical solutions: Use of LMH cell line overexpressing goose - derived RPL7 gene in proliferating goose astrovirus.

[0009] In addition, the present invention discloses a method for promoting the proliferation of goose astrovirus, which infects goose astrovirus into the LMH cell line capable of overexpressing goose - derived RPL7 gene.

[0010] Preferably, the multiplicity of infection of the goose astrovirus is 1.

[0011] Preferably, the LMH cell line is a lentivirus - mediated LMH cell line.

[0012] Preferably, the lentivirus is a lentivirus containing the RPL7 gene.

[0013] In addition, the present invention discloses an LMH cell line, which is an LMH cell line capable of overexpressing goose - derived RPL7.

[0014] In addition, the present invention discloses a preparation method of the above - mentioned LMH cell line, which specifically includes the following steps: Step 1: Extract the RNA of RPL7 from goose tissue cells and reverse - transcribe it into cDNA; Step 2: Refer to the gene sequence of goose - derived RPL7 in NCBI, and use cDNA as a template to amplify the RPL7 gene by PCR with primers RPL7 - F and RPL7 - R; the gene sequence number of RPL7 in GenBank is XM_048077041.2; The gene sequence of the primer RPL7 - F is shown as SEQ ID NO.1, and the gene sequence of the primer RPL7 - R is shown as SEQ ID NO.2; Step 3: Use the RPL7 gene as a template, and further add a Myc - tagged protein at the end of the RPL7 gene by PCR with primers RPL7 - F2 and RPL7 - R - Myc for fusion expression to obtain the target gene RPL7 - Myc; The gene sequence of the primer RPL7 - F2 is shown as SEQ ID NO.3; the gene sequence of the primer RPL7 - R - Myc is shown as SEQ ID NO.4; Step 4: Use the target gene RPL7 - Myc as a template, and perform PCR amplification with homologous - arm primers RPL7 - pLV - F and RPL7 - pLV - R, so that sequences at least about 20 bp long on both sides of the Xba I and Xho I restriction enzyme cleavage sites of the pLV - sfGFP(2A)Puro vector are added to both ends of the RPL7 - Myc gene, and perform homologous recombination with the digested lentiviral vector to construct the plasmid pLV - RPL7; The gene sequence of the primer RPL7-pLV-F is shown in SEQ ID NO.5; the gene sequence of the primer RPL7-pLV-R is shown in SEQ ID NO.6; Step 5: In a cell culture dish, when the monolayer of 293T cells is fused to 90%, transfection is carried out to obtain the pLV-RPL7 recombinant lentivirus suspension; Step 6: Culture LMH cells in good condition, add the pLV-RPL7 recombinant lentivirus suspension and incubate. After incubation, remove the virus solution, add fresh medium and continue culturing and subculturing. After that, add puromycin for resistance screening to obtain the LMH cell line overexpressing RPL7.

[0015] Finally, the present invention also discloses a vaccine, which contains the inactivated goose astrovirus proliferated by the method described above.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The LMH cell line overexpressing goose-derived RPL7 provided by the present invention has a certain effect of promoting the proliferation of goose astrovirus. As a component of the large ribosomal subunit, RPL7 is directly involved in the process of mRNA to protein synthesis. GAstV mRNA interacts with RPL7 and localizes to the endoplasmic reticulum, greatly promoting the reverse transcription level of the virus; RPL7 plays an important role in maintaining the integrity of the virus replication complex, thereby promoting the replication of goose astrovirus; when it is applied to vaccine research and development, it can obtain a higher titer of goose astrovirus, which helps to reduce the cost of vaccine research and development and improve the effect of the vaccine. Description of the Drawings

[0017] Figure 1 is the map of the vector pLV-sfGFP(2A)Puro; Figure 2 is the map of the pLV-RPL7 plasmid; Figure 3 is the agarose gel electrophoresis pattern of the pLV-RPL7 plasmid after double digestion; Figure 4 is the result map of Western Blot for detecting and analyzing the expression level of RPL7-Myc fusion protein in LMH cells; Figure 5 is the result map of RT-qPCR for analyzing the effect of RPL7 overexpression on the mRNA expression level of GAstV in cells; Figure 6 is the result map of Western Blot for analyzing the effect of RPL7 overexpression on the protein expression level of GAstV in cells; Figure 7 is TCID 50Results figure for analyzing the effect of RPL7 overexpression on virus proliferation. Detailed implementation manners

[0018] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0019] Source information of test materials: (1) Cells, viruses and vectors: The chicken liver cancer cell line LMH and the human renal epithelial cell line 293T cells are preserved by the Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture and Rural Affairs of the People's Republic of China and the Key Laboratory of Prevention and Control of Animal-derived Zoonoses of Guangdong Province; The goose astrovirus GAstV GD-ZJ-21-01 strain is preserved by the Institute of Animal Health of Guangdong Academy of Agricultural Sciences / the Key Laboratory of Prevention and Control of Major Poultry Diseases such as Avian Influenza of the Ministry of Agriculture and Rural Affairs of the People's Republic of China; The lentiviral gene overexpression vector pLV-sfGFP(2A)Puro (Cat. VL3408, the full length of the vector is about 9000 bp), its map is as Figure 1 shown, and its supporting lentiviral packaging auxiliary vectors pH1 and pH2 are all products of Beijing Yingmao Shengye Biotechnology Co., Ltd.

[0020] (2) Main reagents and antibodies: The high-fidelity enzyme Phanta® Max Super-Fidelity DNA Polymerase (Cat. P505) and the seamless cloning kit ClonExpress® II One Step Cloning Kit are products of Nanjing Novozymes Biotechnology Co., Ltd.; The restriction enzymes Xba I (R0145) and Xho I (R0146) are products of New England Biolabs; The total RNA extraction kit (Cat. 220011) is a product of Shanghai Feijie Biotechnology Co., Ltd.; The DNA in vitro transfection reagent PolyJet (Cat. SL100688) is a product of SignaGen; The DNA Gel Extraction Kit (Cat. D2500) and the Endo-free Plasmid Midi Kit (Cat. D6929) are products of Omega; The reverse transcription kit PrimeScript RT Master Mix Real Time (Cat. RR036A) is a product of Takara; The fluorescence quantitative PCR enzyme Hieff® qPCR SYBR Green Master Mix (Cat. 11201ES08) is a product of Shanghai Yeasen Biotechnology Co., Ltd.; Both Myc Tag Mouse Monoclonal Antibody (HRP Conjugated) (Cat. AF2867) and GAPDH Mouse Monoclonal Antibody (HRP Conjugated) (Cat. AF2823) are products of Beyotime Biotechnology; The polyclonal antibody against RPL7 (Cat. DF12225) is a product of Affinity; The polyclonal antibody against GAstV was prepared and stored in our laboratory in the early stage. For details, please refer to (Prokaryotic expression of goose astrovirus type 2 VP27 protein and preparation of polyclonal antibody. China Poultry, https: / / link.cnki.net / urlid / 32.1222.S.20231205.1556.004).

[0021] Example 1 (1) Gene amplification and plasmid construction: According to the instructions of the relevant kit, RNA was extracted from goose tissue cells and reverse transcribed into cDNA. Referring to the gene sequence of goose-derived RPL7 in NCBI (GenBank: XM_048077041.2), the RPL7 gene (about 738 bp) was amplified by PCR using the primers (RPL7-F, RPL7-R) designed by ourselves in Table 1 with cDNA as the template; using the recovered RPL7 gene as the template, primers (RPL7-F2, RPL7-R-Myc) were used to further add a Myc tag protein to the end of the RPL7 gene by PCR for fusion expression with the RPL7 gene (RPL7-Myc, 768 bp) for easy detection. The gene sequence of RPL7-Myc is shown in SEQ ID NO.7; atggcggacaaggaagcaaagaagctgccttctgtaccggaaagcctcctgaagaggcggcaggcttatgcagctgcaaaagccaaacgtctgaagaggctgttagctcaaaaaaagtttcgtaaggcacaaagaaaaatcatctatgaaagagccaaagcttaccacaaggagtacaggcacatgtataggcaggagatccgcatggccaggatggcccgaaaagccggcaattactacgttccagctgaacccaagcttgcctttgtgatcaggataagaggcatcaatggtgtcagccccaaggtccgtaaggtgttgcagcttcttcgcctgcgtcagatttttaatggcacattcgtcaagctcaacaaagcttctatcaacatgttgcggattgttgaaccctatattgcatggggttatccaaacctgaagtctgtgcatgagctgatctacaagcgtggttatggcaagatcaacaagaagcgcattgctctgactgataattccctgattcggaaatgccttggaaaacttggcatcatctgcatggaagatgtgatccatgagatttacactgttggcaagaacttcaaagttgtgaacaacttcctttggcccttcaagttatcctctcctcggggtggaatgaagaagaaaacgatccactttgtggagggtggagatgctggtaacagagaagatcagataaacagactcataaggagaatgaacGAGCAGAAACTCATCTCTGAAGAGGATCTG(SEQ ID NO.7)。

[0022] The target gene RPL7-Myc was recovered and used as a template. PCR amplification was performed using homologous arm primers (RPL7-pLV-F, RPL7-pLV-R), so that sequences of at least about 20 bp in length on both sides of the Xba I and Xho I restriction enzyme cleavage sites of the pLV-sfGFP(2A)Puro vector were added to both ends of the RPL7-Myc gene, so that it could undergo homologous recombination with the digested lentiviral vector (double digested with Xba I and Xho I) to construct the plasmid pLV-sfGFP(2A)Puro-RPL7-Myc, hereinafter referred to as pLV-RPL7, and its map reference Figure 2 .

[0023] The result reference Figure 3 , and the results showed that the constructed pLV-RPL7 plasmid was double digested, and the sizes of the gene fragments generated by electrophoresis after digestion were consistent with expectations; further sequencing analysis showed that the above plasmid construction was correct.

[0024] Figure 3 is the agarose gel electrophoresis pattern after double digestion of the pLV-RPL7 plasmid; in the figure, lane 1: undigested pLV-RPL7 plasmid; lane 2: the pLV-RPL7 plasmid was double digested with Xba I and Xho I to generate fragments of about 9000 bp and 783 bp in size; lane 3: DNA marker.

[0025] Table 1 Primer names and sequences

[0026] (2) Lentivirus packaging and infection of LMH cells: In a 10 cm diameter cell culture dish, transfection was carried out when the monolayer of 293T cells was fused to 90%. Take a sterile 1.5 mL centrifuge tube (tube A), add 500 μL of DMEM medium, and then add 20 μL of the DNA in vitro transfection reagent PolyJet and mix well; take another sterile 1.5 mL centrifuge tube (tube B), add 500 μL of DMEM medium, and then add 5 μg of the lentiviral gene overexpression plasmid (pLV-RPL7) to be packaged, 3.75 μg of the lentiviral packaging helper vector pH1 and 1.25 μg of pH2 and mix well. Add the diluted transfection reagent in tube A to tube B, mix well immediately, and incubate at room temperature for 10 - 15 min. While waiting for incubation, digest 293T cells with trypsin and prepare them into about 6×10 5 cells·mL -1Cell suspension. According to the amount of virus to be prepared, place the cell suspension in a 15 mL or 50 mL centrifuge tube for standby; add 1 mL of transfection mixture to 10 mL of cell suspension, gently pipette to mix the cells; add the mixed cell suspension to a 10 cm cell culture dish and culture at 37 °C and 5% CO2 for 24 h; remove the medium containing the transfection reagent, wash the cells twice, and add 10 mL of lentivirus medium (DMEM medium + 10% FBS + 1 mM sodium pyruvate). After 48 h, collect the supernatant of the cell culture medium, centrifuge at 500 g for 10 min at 4 °C to remove cell debris; the recombinant lentivirus particles carrying the gene to be expressed are contained in this supernatant and can be directly used to infect LMH cells.

[0027] (3)Construction and identification of LMH cell line stably overexpressing RPL7: Evenly inoculate healthy LMH cells into a 24-well plate for culture. When the cells grow to 70% - 80%, discard the culture medium, add the pLV-RPL7 recombinant lentivirus suspension and incubate at 37 °C (set a blank control group at the same time); after incubating for 2 h, discard the virus solution, add fresh medium and continue to culture and passage; add puromycin with a final concentration of 2 μg / mL for resistance screening at 48 - 72 h; the obtained positive clone is the LMH cell line overexpressing RPL7 mediated by lentivirus, named LMH-RPL7, and the control group is LMH-NC.

[0028] It can be seen from Figure 4 that the RPL7-Myc fusion protein was successfully overexpressed in LMH-RPL7 cells, while the expression of this fusion protein was not detected in the control group of LMH-NC cells. On the other hand, detection using the RPL7 polyclonal antibody can also find that the expression level of RPL7 protein in LMH-RPL7 cells is significantly higher than that in the control group of LMH-NC, indicating that the construction of the LMH cell line stably overexpressing RPL7 was successful.

[0029] Figure 4 It is the result diagram of Western Blot detection and analysis of the expression level of RPL7-Myc fusion protein in LMH cells.

[0030] Example 2 Analysis of the promoting effect of LMH cell line overexpressing RPL7 on the proliferation of GAstV LMH-RPL7 and LMH-NC cells were infected with GAstV at a multiplicity of infection (MOI) of 1 and incubated in a 37 °C, 5% CO₂ incubator for 2 h for adsorption. After adsorption, the inoculum was discarded, and the cells were washed 3 times with PBS and then cultured in maintenance medium. Cells were harvested at different time points of 24 h and 48 h post-infection to extract cellular proteins and RNA. The replication level of GAstV in the cells was analyzed by Western Blot and RT-qPCR (primers: 5’-GGGTGATCCGCAAGGAAATA-3’, 5’-AAGTTTCGCCAGGGTTAGAG-3’). Meanwhile, the cell supernatant virus solution was collected, and the virus solution was repeatedly frozen and thawed three times. After centrifugation, the supernatant was taken, and the supernatant was serially diluted 10-fold and used to infect LMH cells in a 96-well plate. After virus infection, the cells were maintained for 3 d, and the positivity or negativity of GAstV in each cell well was determined by RT-qPCR. The virus titer in each supernatant was measured by the Reed-Muench method.

[0031] The results can be seen from Figure 5 , Figure 6 and Figure 7 that in the LMH cell line overexpressing the RPL7 gene, whether it is the mRNA expression level, protein expression level of the virus in the cells, or the virus titer in the cell supernatant, they are all significantly higher than those in LMH-NC cells. The above indicates that the LMH cell line overexpressing the RPL7 protein has a significant promoting effect on the proliferation of GAstV.

[0032] Figure 5 Figure showing the results of RT-qPCR analysis of the effect of RPL7 overexpression on the mRNA expression level of GAstV in cells; Figure 6 Figure showing the results of Western Blot analysis of the effect of RPL7 overexpression on the protein expression level of GAstV in cells; Figure 7 Figure showing the results of TCID 50 analysis of the effect of RPL7 overexpression on virus proliferation.

[0033] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be encompassed within the present invention.

Claims

1. Use of LMH cell line overexpressing goose-derived RPL7 gene in proliferating goose astrovirus.

2. A method for promoting the proliferation of goose astrovirus, characterized in that, Infect goose astrovirus into the LMH cell line capable of overexpressing goose-derived RPL7 gene.

3. The method according to claim 2, characterized in that, The multiplicity of infection of the described goose astrovirus is 1.

4. The method according to claim 2, wherein The described LMH cell line is a lentivirus-mediated LMH cell line.

5. The method according to claim 4, wherein The described lentivirus is a lentivirus containing the RPL7 gene.

6. A LMH cell line, characterized in that, The described LMH cell line is an LMH cell line overexpressing goose-derived RPL7.

7. The method for preparing the LMH cell line according to claim 6, wherein, Specifically, it includes the following steps: Step 1: Extract the RNA of RPL7 from goose tissue cells and reverse transcribe it into cDNA; Step 2: Refer to the gene sequence of goose-derived RPL7 in NCBI, and use cDNA as a template to amplify the RPL7 gene by PCR with primers RPL7-F and RPL7-R; the gene sequence of RPL7 is numbered XM_048077041.2 in GenBank; The gene sequence of the primer RPL7-F is shown as SEQ ID NO.1, and the gene sequence of the primer RPL7-R is shown as SEQ ID NO.2; Step 3: Use the RPL7 gene as a template, and further add a Myc tag protein to the end of the RPL7 gene by PCR with primers RPL7-F2 and RPL7-R-Myc for fusion expression to obtain the target gene RPL7-Myc; The gene sequence of the primer RPL7-F2 is shown as SEQ ID NO.3; the gene sequence of the primer RPL7-R-Myc is shown as SEQ ID NO.4; Step 4: Use the target gene RPL7-Myc as a template, and perform PCR amplification with homologous arm primers RPL7-pLV-F and RPL7-pLV-R, so that sequences at least 20 bp long on both sides of the Xba I and Xho I restriction enzyme cleavage sites of the pLV-sfGFP(2A)Puro vector are added to both ends of the RPL7-Myc gene, and perform homologous recombination with the digested lentiviral vector to construct the plasmid pLV-RPL7; The gene sequence of the primer RPL7-pLV-F is shown as SEQ ID NO.5; the gene sequence of the primer RPL7-pLV-R is shown as SEQ ID NO.6; Step 5: In a cell culture dish, when the 293T cell monolayer is fused to 90%, perform transfection to obtain the pLV-RPL7 recombinant lentivirus suspension; Step 6: Culture the LMH cells in good condition, add the pLV-RPL7 recombinant lentivirus suspension for incubation, remove the virus solution after incubation, add a new culture medium to continue culturing and passaging, and add puromycin for resistance screening after completion to obtain the LMH cell line overexpressing RPL7.

8. A vaccine, characterized in that, The described vaccine contains inactivated goose astrovirus proliferated by the method described in any one of claims 2-5.

Images