CN116751266A - Broad-spectrum henipav protective antigen and application thereof - Google Patents

Broad-spectrum henipav protective antigen and application thereof Download PDF

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CN116751266A
CN116751266A CN202310785283.4A CN202310785283A CN116751266A CN 116751266 A CN116751266 A CN 116751266A CN 202310785283 A CN202310785283 A CN 202310785283A CN 116751266 A CN116751266 A CN 116751266A
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virus
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陈薇
李耀辉
徐俊杰
李汭桦
宰晓东
黄小燕
张军
刘树玲
宋小红
杨益隆
殷瑛
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Academy of Military Medical Sciences AMMS of PLA
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Abstract

The invention discloses a Hendela virus G protein mutant, which is characterized in that serine at 586 th position of a Hendela virus receptor binding (G) protein is mutated into asparagine. Compared with wild type antigen, the mutant antigen can better excite the broad-spectrum neutralizing antibody reaction. The mutant antigen is combined with AL (OH) 3 Equal vaccine adjuvant or carried in 5 typeAfter adenovirus vector, can obviously promote the cross antibody reaction of host against Nipah virus, and can be used for the universal henpah virus vaccine with broad-spectrum protection effect. The invention also provides application of the mutant antigen in preparation of henipa virus vaccine.

Description

Broad-spectrum henipav protective antigen and application thereof
Technical Field
The invention discloses a protective antigen and a nucleic acid molecule, and belongs to the technical field of polypeptides and nucleic acids.
Background
Hendra virus (Hendra virus, heV) and Nipah virus (NiV) belong to the genus hennipah virus of the family paramyxoviridae, and are a single-stranded negative strand RNA virus. The natural host of Henipavirus (HNV) HNV is bata of the family foxidae, which is very geographically widespread, and HNV has been shown to spread between humans, thus risking pandemic. Nipah virus was first shown in Malaysia in 1998 and then periodically outbreaks in south Asia, resulting in over 600 infections with a mortality rate of 40-100%. Hendra virus type I was first discovered in 1994 in hendra town in eastern australia, resulting in tens of deaths, and was isolated in 1996 in fruit bats in australia, and later exploded in eastern australia multiple times. Type II hendra virus 2022 was isolated in horses that died from infection with HeV in queensland, australia. The amino acid homology of the G protein of the type I and type II hendra viruses is 92.5 percent, and the two have similar structure and function. The 1-71 amino acids of Hendela virus G protein are N-terminal intracellular region and transmembrane region, and the 72-186 amino acids are "stem" domain, which has the functions of supporting head region and forming polymer, and the 187 amino acids to the end of C-terminal are "head" domain of binding receptor composed of 6 beta sheet.
WHO in 2018 classified nipah virus disease caused by nipah virus and other hennipah virus diseases as one of 10 potentially high-risk infectious diseases that require major attention. Vaccines are critical for preventing HNV outbreaks, more than 40 henipav virus vaccines are currently under development, but no human HNV vaccine has been marketed as a batch to date. Nipah and hendra viruses use G proteins, which are the primary targets in current nipah and hendra virus vaccine development, to bind to cell receptors ephrinB2 and ephrinB3 to initiate infection of cells.
The amino acid homology of the G proteins of the Nipah virus and the Hendela virus is about 79%, and the development of a general vaccine with protective effect on the two viruses is of great importance. Ma Yongheng Deltavirus vaccine against soluble Hendela virus G protein has been approved for use in Australia. This candidate vaccine has also been shown to have protective effects against nipah virus challenge in non-human primates, and has entered a phase of clinical trials. There are also studies that have found that only limited cross-antibody responses were elicited after immunization of mice with the G proteins of hendra and nipah viruses, both of which induced cross-antibody titers that were more than 100-fold lower than autoantibody titers, and also only limited cross-protective antibody responses were elicited, suggesting that the use of only one G protein as an antigen may not produce adequate protection for both viruses.
The invention aims to improve the capability of exciting cross antibody reaction against Nipah virus by modifying wild Hendela virus antigen, thereby obtaining the universal vaccine antigen of Nipah virus and Hendela virus with broad-spectrum protection effect.
Disclosure of Invention
In view of the above, the present invention provides a hendra virus G protein mutant comprising a sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2 from serine to asparagine, wherein the amino acid sequence of SEQ ID NO:1 is the wild type sequence of the hendra virus type I G protein, SEQ ID NO:2 is the wild type sequence of the type II hendra virus G protein. The Hundela virus G protein mutant is obtained by mutating the 586 th amino acid of the full-length sequence (604 amino acids) of wild type I and II type Hundela virus G protein from serine to asparagine, and the mutation is named as S586N, so that the Hundela virus G protein mutant disclosed by the invention is the Hundela virus G protein containing the mutation site of S586N.
In a preferred embodiment of the present invention, the amino acid sequence of the hendra virus G protein mutant is as set forth in SEQ ID NO: 3. The mutant is obtained by intercepting a mutant with an amino acid sequence shown as SEQ ID NO:1 and contains amino acids 187-604 of the type I hendra virus G protein, and a "S586N" mutation site, said mutant being designated "HNV-G1". The truncated hendra virus G protein was designed to increase the solubility of the recombinant protein mutants.
Secondly, the invention provides application of the hendra virus G protein mutant in preparing henipavirus disease vaccine or therapeutic drugs. In the invention, the hendra virus G protein mutant can be used as a protective antigen to prepare a vaccine for preventing henipavirus diseases. Vaccine prepared with HNV-G1 as protective antigen immunized 6-8 week old female BALB/c mice with a mean NiV neutralizing antibody titer of 3.73 (log 10), respectively, significantly higher than the HeVG immunized group (log 10 mean neutralizing antibody titer of 2.47). Wherein the average neutralizing antibody titer of NiV is 17.9 times that of the HEVG immune group, and HNV-G1 can be proved to greatly improve the cross neutralizing antibody level of antigen excitation. Those skilled in the art will appreciate that the same immune effect as HNV-G1 can be achieved by including the "S586N" mutation site, based on the full length of the Hendela virus G protein mutant, as well as on other truncated lengths of Hendela virus G protein that increase the solubility of the recombinant protein.
Third, the present invention provides a nucleic acid molecule encoding the above-described hendra virus G protein mutant. By using the nucleic acid molecule provided by the invention, the recombinant protein of the Hendela virus G protein mutant can be obtained by a genetic engineering technology.
In a preferred embodiment of the invention, the nucleic acid molecule has the sequence set forth in SEQ ID NO:4 from nucleotide 109-1365. As is well known to those skilled in the art, the aforementioned Hendela virus G protein mutants may also be obtained without altering the amino acid sequence, using other nucleic acid molecules encoding the aforementioned Hendela virus G protein mutants. Thus, all the sequences encoded as SEQ ID NOs: 1, and all fall within the scope of the present invention.
Fourth, the present invention provides an expression vector containing the above nucleic acid molecule. The recombinant protein of the Hendela virus G protein mutant can be obtained by utilizing the expression vector through conventional genetic engineering technology.
Fifth, the present invention provides a host cell expressing the above-described hendra virus G protein mutant, which contains the above-described expression vector.
In a preferred embodiment of the present invention, the expression vector is pcDNA3.1 eukaryotic expression vector, recombinant proteins of the Hendela virus G protein mutant are obtained by an Expi293F mammalian cell expression system, and it is expected by those skilled in the art that Hendela virus G protein mutants of the present invention can also be obtained by eukaryotic expression systems, such as CHO cells, using other eukaryotic expression vectors, as long as the Hendela virus G protein mutant-encoding nucleic acid provided by the present invention is used.
Sixth, the invention provides a recombinant human adenovirus vector carrying nucleic acid encoding a hendra virus G protein mutant, wherein the amino acid sequence of the hendra virus G protein mutant is shown as SEQ ID NO:6. the recombinant human adenovirus vector enters a host cell and can express an amino acid sequence shown as SEQ ID NO:6, a hendra virus G protein mutant.
The mutant is obtained by intercepting a mutant with an amino acid sequence shown as SEQ ID NO:1 and contains amino acids 72-604 of the type I hendra virus G protein and a "S586N" mutation site, and the recombinant human adenovirus vector is named "Ad5-HNV-G1". The truncated hendra virus G protein was designed to increase the solubility of recombinant protein mutants expressed by recombinant human adenovirus vectors.
In a preferred embodiment of the present invention, the recombinant human adenovirus vector is provided with a nucleic acid encoding a tPA signal peptide and a nucleic acid encoding a flexible connecting peptide at the 5' end of the nucleic acid encoding the hendra virus G protein mutant.
In a more preferred embodiment of the present invention, the nucleic acid encoding tPA signal peptide, the nucleic acid encoding flexible connecting peptide, and the nucleic acid encoding the hendra virus G protein mutant carried in the recombinant human adenovirus vector are in tandem to form a nucleic acid molecule having a sequence as set forth in SEQ ID NO: shown at 7.
Finally, the invention provides application of the recombinant human adenovirus vector in preparing henipa virus vaccine or therapeutic medicine. The recombinant human adenovirus vector is used for loading the Hendela virus G protein mutant coding nucleic acid as a vaccine to immunize an experimental mouse through an injection way, so that the mouse obtains excellent humoral immunity and cellular immunity, and therefore the recombinant human adenovirus vector can be prepared into a vaccine for preventing Hendela virus diseases, and can also be used as a medicament under the condition of emergency infection so as to enable a patient to timely obtain the humoral immunity and the cellular immunity to the Hendela virus diseases.
The recombinant human adenovirus vector is used for loading the Hendela virus G protein mutant coding nucleic acid as a vaccine to immunize an experimental mouse through an injection way, so that the mouse obtains excellent humoral immunity and cell immunity. It is contemplated by those skilled in the art that the above-described immune effects can be obtained using other vaccine vectors, as long as the nucleic acid encoding the hendra virus G protein mutant provided by the present invention, which contains the "S586N" mutation site, is used, whether based on the full length of the hendra virus G protein mutant, and based on other truncated lengths of the hendra virus G protein that enhance the solubility of the recombinant protein. Expression vectors containing the nucleic acid molecules described above may also be delivered into humans by in vivo delivery techniques, with the technical effect of immunization achieved by expression in vivo. The nucleic acid molecule delivered may be a DNA molecule or an mRNA molecule. The delivery route can be direct intramuscular injection, intravenous injection, subcutaneous injection, stereoscopic in vivo injection, or can be administration route such as liposome transfection, nanoparticle carrier delivery and the like.
When the mutant with the mutation from serine 586 to asparagine of the receptor binding (G) protein of the hendra virus provided by the invention is used as a protective antigen, compared with a wild-type antigen, the mutant can better excite a broad-spectrum neutralizing antibody reaction. The mutant antigen is combined with AL (OH) 3 After the vaccine adjuvant or the 5-type adenovirus vector is loaded, the cross antibody reaction of a host against the Nipah virus can be obviously improved, and the vaccine adjuvant can be used for a universal henpah virus vaccine with a broad-spectrum protection effect.
Drawings
FIG. 1 evaluation of mean neutralizing antibodies to NiV and HeV stimulated by HNV-G1 recombinant proteins;
FIG. 2 evaluation of neutralizing antibody levels against two genotypes of NiV-G induced by the Ad5-HNV-G1 vaccine group;
FIG. 3 evaluation of the level of cellular immunity induced by the Ad5-HNV-G1 vaccine group.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are only exemplary and do not limit the scope of the invention in any way, which is defined by the claims.
Example 1 preparation of HNV-G1 antigen
1.1 Construction of HNV-G1 and HeV-G protein expression plasmids
The mutant Hendela virus G protein mutant carrying the S586N mutation is named as HNV-G1, and the HNV-G1 has the amino acid sequence shown in SEQ ID NO:1, and on the basis of a mutant of the I-type Hendela virus G protein in which the 586 th amino acid is mutated from serine to asparagine, intercepting the 187 th to 604 th amino acids, wherein the amino acid sequence of HNV-G1 is shown as SEQ ID NO: 3.
Meanwhile, the wild Hendela virus G protein is used as a control, and the amino acid sequence of the Hendela virus G protein is shown as SEQ ID NO:1 (without the "S586N" mutation site). Codon optimization is carried out on genes encoding HNV-G1 antigen (187-604 AA) and wild Hendela virus G protein (187-604 AA), and the codon sequence of the HNV-G1 after optimization is shown as SEQ ID NO:4, and the optimizing target species is human. Synthesizing the optimized gene sequence and constructing a pcDNA3.1 eukaryotic expression vector, sequentially adding a kozak sequence (CGCCACC), an initiation codon ATG, a secretory expression tPA signal peptide MDAMKRGLCCVLLLCGAVFVSNS and a Strep tag WSHPQFAK in front of a coding region, and obtaining a nucleic acid full-length sequence (tPA signal peptide coding sequence+strep tag+GGGGS coding sequence+HNV-G1 codon optimization sequence) with the sequence shown in SEQ ID NO:4, wherein the tPA signal peptide has a coding sequence shown in SEQ ID NO:4, and the coding sequence of the Strep tag is shown as SEQ ID NO:4, and the GGGGS coding sequence is shown as SEQ ID NO:4 from nucleotide 94 to 108. Meanwhile, HEV-G of wild type Bengalia Nipah virus G protein is constructed, the amino acid sequence of HEV-G is identical to HNV-G1 except that the amino acid sequence does not contain S586N mutation site, and the codon optimization sequence of encoding HEV-G is shown as SEQ ID NO: shown at 5. Also preceding (5' end) the HEV-G coding region is a tandem tPA signal peptide coding sequence +strep tag +GGGGS coding sequence, which is identical to the sequence of SEQ ID NO:4 are identical at nucleotides 1-108. Gene synthesis and vector construction were accomplished by general biosystems (Anhui) Inc.
1.2 Expression and purification of HNV-G1 proteins
The G protein was expressed using an Expi293F mammalian cell expression system.
1) Culturing and expansion of the Expi293F suspension cells were performed. Cell density was adjusted to 3×10 per ml using an Expi293 expression medium prior to transfection 6 And each.
2) 30 microgram of G protein expression plasmid was prepared and 30 milliliters of cells were transfected using the Expi293 transfection kit.
3) The transfected cells are placed in a shaking table for culture, the culture condition is 120rpm, the relative humidity is more than or equal to 80%, and the carbon dioxide concentration is 8%.
4) After 72 hours of transfection, the cell culture broth was taken and centrifuged at 3000g for 15min. The supernatant was filtered using a 0.45 μm syringe filter to remove cell debris. The filtered supernatant was used for subsequent protein purification.
Purification was performed using Strep-trap affinity column proteins, as follows:
1) The column was equilibrated with 3-5 column volumes of equilibration buffer (20 mM Tris-HCL,20mM NaCl,1mM EDTA) over AKTA protein purification.
2) Loading after the UV280 curve is gentle, and balancing by using a balancing buffer solution after loading.
3) The column volumes were equilibrated for 5-10 volumes, eluted with elution buffer (2.5mM desthiobiotin,20mM Tris-HCL,20mM NaCl,1mM EDTA) after the UV280 curve was gentle and the elution peaks were collected.
4) The eluate was exchanged and concentrated using a 50kDa cut-off ultrafiltration tube, the buffer was exchanged for PBS, and the centrifugation was performed three times at 3000g, 4℃for 15min.
5) Protein concentration was determined using BCA kit (Pierce ™ BCA Protein Assay kit).
Example 2 immunological evaluation of HNV-G1 antigen on mouse model
2.1 Immunization of animals
Female BALB/c mice of 6-8 weeks of age were selected, 6 per group. The purified G protein and mutant are dissolved in PBS, aluminum hydroxide adjuvant and CpG1826 adjuvant are added, and the mixture is mixed for 8 hours at 4 ℃ in a rotating way, wherein the dose of each mouse immunization is 10 mug protein, 200 mug aluminum hydroxide adjuvant and 20 mug CpG1826 adjuvant. Control mice were injected with 100 μl PBS per mouse. Primary immunization was performed on day 0 and booster immunization was performed on day 21. Collecting 42 days after immunization, collecting blood by tail vein blood sampling method, standing serum at room temperature for 4 hr, centrifuging 8000g for 15min after blood delamination, and transferring serum into new centrifuge tube. Mice were sacrificed 42 days after immunization using carbon dioxide asphyxiation.
2.2 Henipa virus neutralizing antibody detection
The main method for detecting the neutralizing antibody of the Nipah virus, namely a liquid phase chip, is used for detecting the neutralizing antibody reaction excited by the immunity of the recombinant protein vaccine. The isolated mouse serum was placed in a black opaque 96-well plate, and magnetic microspheres coated with nipah virus and hendra virus G protein were added to the culture plate, 1500 per magnetic bead. Dilute biotin-conjugated ephrin-B2 receptor was added and incubated with shaking at 800rpm for 60 minutes. The streptavidine-r-phyreythrin (SAPE) was added and incubated with shaking at 800rpm for 30 minutes. The supernatant was aspirated using a magnetic plate separator, washed three times with PBS (1% bsa), and finally the binding was determined using a Luminex magix liquid phase chip detector.
As shown in FIG. 1, the results demonstrate that HNV-G1 stimulated NiV average neutralizing antibody titers were 3.73 (log 10), respectively, significantly higher than the HEVG immunized group (log 10 average neutralizing antibody titers of 2.47). Wherein the NiV average neutralizing antibody titer was 17.9 times that of the HeVG immunized group. This suggests that HNV-G1 could greatly increase antigen-stimulated cross-neutralizing antibody levels. Meanwhile, the average neutralizing antibody titer of HNV-G1-stimulated HeV was 3.86 (log 10), which was not significantly different from that of the HeVG immunized group (3.61).
Example 3 preparation of AD5-HNV-G1 adenovirus vector vaccine
Intercepting a Hendela virus (Gene ID: 1446471) G glycoprotein mutant (SEQ ID NO:1 containing an S586N mutation site), carrying out sequence optimization on 72-604 th amino acid segments (the sequences of the segments are shown as SEQ ID NO: 6), adding a coding sequence of a tPA signal peptide MDAMKRGLCCVLLLCGAVFVSNS at the 5' end of the optimized sequence, and taking the coding sequence as a coding sequence of a GGGGS flexible polypeptide of a connector, wherein the full-length sequence of nucleic acid (tPA signal peptide coding sequence+GGGGS coding sequence+HNV-G1 codon optimization sequence) after tandem is shown as SEQ ID NO:7, wherein, 1-69 are tPA signal peptide coding sequence, 70-84 are GGGGS coding sequence, 85-1686 are HNV-G1 codon optimization sequence. The recombinant human adenovirus vector containing the above tandem sequence was named "AD5-HNV-G1". Meanwhile, the Ad5-HEV-G of the wild Hendela virus G protein is constructed, and other sequences are the same except that the S586N mutation site is not contained, wherein the adenovirus packaging and detection work is carried out by ABM (Applied Biological Materials) biotechnology (China) (the construction method is shown in Danthinne, X., & Imperiale, M.J. (2000) & Production of first generation adenovirus vectors: a review, gene therapy, 7 (20), 1707-1714, https:// doi.org/10.1038/sj.gt.3301301). The adenovirus obtained was subjected to titer determination using an end point dilution method.
EXAMPLE 4 immunological evaluation of AD5-HNV-G1 adenovirus vector vaccine on mouse model
4.1 Immunization of animals
Vaccine immunization experiments were performed using female BALB/c mice 6-8 weeks old without specific pathogen. 6 mice per group, each immunized 1X 10 7 pfu Ad5-HNV-G1, control group immunized 1×10 7 Ad5-HEV-G carrying wild-type Hendela virus G protein in pfu was subjected to Gene codon optimization using the same method and recombinant adenovirus vector vaccine Ad5-NIV-G carrying wild-type Bengalia virus G protein Gene (Gene ID: 920955) was constructed, and immunization was performed as a control, and 1X 10 mice were immunized 7 pfu, blank group were immunized with PBS and all mice were given 100 μl by intramuscular injection.
4.2 Henipa virus neutralizing antibody detection
The detection of Ad5-NiV immune-elicited neutralizing antibody reactions was performed using a mainstream method for detecting nipah virus neutralizing antibodies, liquid phase chip. The isolated mouse serum was placed in a black opaque 96-well plate, and magnetic microspheres coated with nipah virus and hendra virus G protein were added to the culture plate, 1500 per magnetic bead. Dilute biotin-conjugated ephrin-B2 receptor was added and incubated with shaking at 800rpm for 60 minutes. The streptavidine-r-phyreythrin (SAPE) was added and incubated with shaking at 800rpm for 30 minutes. The supernatant was aspirated using a magnetic plate separator, washed three times with PBS (1% bsa), and finally the binding was determined using a Luminex magix liquid phase chip detector.
The experimental results are shown in FIG. 2, the titres of neutralizing antibodies induced by the Ad5-HNV-G1 vaccine group against the two genotypes NiV-G were 3.00 and 2.89 (log 10), respectively, which are significantly higher than those of the Ad5-HeV-G vaccine group (2.31 and 2.25), whereas the titres of neutralizing antibodies against the two HeV-G strains Ad5-HNV-G1 were 3.20 and 3.21, and the titres of Ad5-HeV-G were 3.23 and 3.19, respectively, which were not significantly different (FIG. 2).
4.3 Detection of cellular immune response using enzyme-linked immunodot blot assay (ELISpot)
1) Enclosed ELISPot plates: the ELISPot plate was washed three times with 100. Mu.L/well of sterile PBS, and finally 10% FBS RPMI1640 100. Mu.L/well was added and incubated for 30 min at room temperature.
2) Spleen cells and stimulus were added: spleen cells were isolated and counted as before, and finally added 2.5X10 5 cells/well, overlap peptide pool (1. Mu.g/mL single peptide concentration) was added, positive control wells added with stimulus PMA+deionized mycin mix, negative control wells added with equal volume of cell culture medium.
Wherein the overlapping peptide library is prepared as follows: hendela virus (Gene ID: 1446471) has 604 amino acids in the entire G glycoprotein, and we have selected the extracellular domain of the G protein, i.e., from amino acids 71-604, for peptide library synthesis. 15 amino acids were selected as a peptide stretch, with a step size of 4, and 11 overlaps for each peptide stretch. 130 peptides were synthesized in total and all peptides were desalted (synthesized by Jier Biochemical Co., ltd.) to construct a T cell epitope peptide library. The T cell epitope peptide can specifically stimulate immune cells, does not need internalization and processing of APC, can be directly presented to the T cells, each T cell epitope peptide has corresponding MHC molecules, represents a specific cellular immunity, and a plurality of T cell epitope peptides are mixed to form a T cell epitope peptide library or peptide pool. Stimulation with a peptide pool is effective to elicit cytokine secretion from specific T cells, thereby detecting the level of specific cellular immunity elicited by the candidate vaccine.
3) Stimulation culture: the ELISPot plates were placed into a cell incubator at 37℃for incubation 40-48 h.
4) Cleaning the plate: after the incubation, the culture medium in the culture plate is scattered, PBS 200 mu L/well is added for cleaning ELISPOT5 times, and the culture medium can be inversely buckled on water absorption paper to remove liquid in the well as much as possible.
5) Incubation resistance: the detection antibody (R4-6A 2-biotin) was diluted to 1. Mu.g/mL using PBS, 100. Mu.l of antibody dilution was added to each well, and incubated at room temperature for 2 hours.
6) Cleaning the plate: repeating the step (4).
7) Secondary antibody incubation: secondary antibody (strepavidin-HRP) was diluted with PBS 1:1000, 100 μl of secondary antibody dilution was added and incubated for 1 hour at room temperature.
8) Cleaning the plate: repeating the step (4).
9) Color development and reading: adding 100 mu L/hole of the color development liquid, placing the color development liquid in a incubator at 37 ℃ in a dark place for color development for about 6-10 minutes, taking out the plate after spots are clear, and flushing the plate with pure water to terminate the color development. And (5) after the plates are dried, placing the plates in an ELISPot spot counter for counting.
The ELISPot results are shown in FIG. 3, and the results show that spleen cells of mice in three adenovirus vector vaccine groups can be stimulated by HeV-G1-G and HeV-G2-G to generate IFN-gamma, wherein the average IFN-gamma secretion cell spot number of the Ad5-HNV-G1 group after NiV-G stimulation is 314.8, the Ad5-NiVG group is 250.3, and the Ad5-HeVG group is 301.8; the average IFN-gamma secretion cell spot number of the Ad5-HNV-G1 group after the HeV-1-G stimulation is 2005.2, the Ad5-NiVG group is 201.3, the Ad5-HeVG group is 1783.5, the average IFN-gamma secretion cell spot number of the Ad5-HNV-G1 group after the HeV-2-G stimulation is 2829.8, the Ad5-NiVG group is 530.3, and the Ad5-HeVG group is 2320.8 (FIG. 3). The above results indicate that Ad5-HNV-G1 is effective in eliciting a broad spectrum of cellular immune responses against NiV and HeV.

Claims (10)

1. A hendra virus G protein mutant, wherein the hendra virus G protein region mutant comprises an amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2 from serine to asparagine.
2. The hendra virus G protein mutant of claim 1, wherein the amino acid sequence of the hendra virus G protein is set forth in SEQ ID NO: 3.
3. Use of a hendra virus G protein mutant according to claim 1 or 2 for the preparation of a henipa virus disease vaccine or therapeutic.
4. A nucleic acid encoding a mutant hendra virus G protein of claim 2, wherein the nucleic acid has a sequence set forth in SEQ ID NO: 4.
5. An expression vector comprising the nucleic acid of claim 4.
6. A host cell expressing the hendra virus G protein mutant of claim 1 or 2, wherein said host cell comprises the expression vector of claim 5.
7. A recombinant human adenovirus vector comprising a nucleic acid encoding a mutant hendra virus G protein of claim 1, wherein said mutant hendra virus G protein has an amino acid sequence set forth in SEQ ID NO:6.
8. the recombinant human adenovirus vector according to claim 7, wherein the recombinant human adenovirus vector is provided with a nucleic acid encoding a tPA signal peptide and a nucleic acid encoding a flexible linker peptide at the 5' end of the nucleic acid encoding the hendra virus G protein mutant.
9. The vector of claim 8, wherein the nucleic acid encoding tPA signal peptide, the nucleic acid encoding flexible connecting peptide, and the nucleic acid encoding the hendra virus G protein mutant carried in the recombinant human adenovirus vector are in tandem with the nucleic acid molecule having the sequence set forth in SEQ ID NO: shown at 7.
10. Use of the recombinant human adenovirus vector of claim 9 in the preparation of a henipav virus disease vaccine or therapeutic.
CN202310785283.4A 2023-06-29 2023-06-29 Broad-spectrum henipav protective antigen and application thereof Pending CN116751266A (en)

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