CN113980100B

CN113980100B - Adenovirus vector recombinant new coronavirus B.1.429 variant vaccine and application thereof

Info

Publication number: CN113980100B
Application number: CN202111586045.8A
Authority: CN
Inventors: 陈薇; 王步森; 侯利华; 徐婧含; 吴诗坡; 张哲�; 赵拯浩; 张金龙; 宋小红; 王玉东; 付玲; 陈旖
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2022-07-12
Anticipated expiration: 2041-12-23
Also published as: CN113980100A

Abstract

The invention discloses a new crown variant vaccine taking human 5-type replication-defective adenovirus as a vector. The vaccine takes replication-defective human adenovirus type 5 with combined deletion of E1 and E3 as a vector, and a novel coronavirus B.1.429 variant antigen gene (Ad 5-nCoV-B.1.429) which is optimally designed is integrated in a genome. The vaccine can effectively express protective antigen protein in host cells. The vaccine can be used for stimulating antibody reaction against a new crown wild strain and B.1.351 and B.1.617.2 variant strains by single immunization. When the vaccine is used together with a 2019 wild-type novel coronavirus vaccine, strong and broad-spectrum neutralizing antibody reaction of a novel coronavirus variant strain can be excited after the vaccine is boosted. The vaccine can excite a broad-spectrum neutralizing antibody reaction when being used as independent immunity or used as heterotype strengthening immunity together with a new crown wild strain vaccine, has certain application advantages, can be used as a vaccine candidate strain, and is used for coping with the epidemic situation of a continuously spread new crown variant strain.

Description

Adenovirus vector recombinant new coronavirus B.1.429 variant vaccine and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant novel coronavirus B.1.429 variant vaccine.

Background

Since the discovery of new original strains of coronavirus in the early 2020, the variants emerge in endlessly. Because the new coronavirus is an RNA virus, the long-chain RNA genome structure of the new coronavirus is easy to mutate or recombine genes in the evolution process, thereby generating a variant strain. The long circulation and spread of RNA viruses among hosts increases the likelihood of emergence of immune escape variants and even "superstrains". By 11 months 2021, the b.1.1.7 (Alpha), b.1.351 (Beta), p.1 (Gamma) and b.1.617.2 (Delta) variant viruses were classified as "Variants of interest" Strains (VOCs) by the world health organization or the american center for disease prevention and control. In addition, B.1.429 (Epsilon), which originates in the United states, has also been included in "VOC" virus strains, and as the prevalence and dominance of the above-mentioned 4 variants have increased, the interest in B.1.429 variants has decreased.

Among the above "VOC" mutants, the b.1.1.7 variant had the least effect on the recovery period and neutralizing antibodies after vaccination; the P.1 variant showed moderate neutralization resistance with approximately 3-7 fold reduction in neutralizing antibody titers; the B.1.351 variant increased neutralizing resistance to convalescent and vaccinated serum samples, approximately 7-42 fold lower; the mRNA vaccines BNT162b2 and mRNA-1273 were reduced by about 2-3 times against the B.1.617.2 variant. Consistent with the decreased level of neutralizing antibodies, the protective efficacy of various vaccines has decreased significantly in variant endemic areas. The recombinant protein vaccine NVX-CoV2373 was 96% effective in the UK against COVID-19 caused by wild-type SARS-CoV-2 strain, whereas in HIV-negative test participants in south Africa, the effectiveness was reduced to 51%, with the majority of the infectors being B.1.351 variants. In catal, BNT162b2 was 95% effective against the wild strain, while the b.1.1.7 variant was about 90%, the b.1.351 variant was about 75%, and the b.1.617.2 variant was 51.9%. Cov2.s is 72% effective in moderate to severe disease in the united states (mainly wild-type strains), and drops to 52% in south africa, with about 94.5% of cases infected with b.1.351 variants. The ChAdOx1 nCoV-19 vaccine has 84% protective efficacy against wild strains in the UK and 75% protective efficacy against B.1.1.7 variants, but only 10% protective efficacy against mild to moderate disease in the regions of south Africa where B.1.351 variants are prevalent.

The b.1.429 variant showed moderate neutralizing resistance to the convalescent and vaccinated serum samples. Compared with other variants, the spike protein has the least mutation sites, including the L452R site located in the receptor binding region, the D614G site located in the S1 region, and the S13I and W152C sites located in the S1 NTD. Among them, D614G is ubiquitous in B.1.1.7, B.1.351, P.1 and B.1.617.2 epidemic dominant strains, and L452R site is coexistent in B.1.617.1 (Kappa) and B.1.617.2 variant strain with late coverage rate close to 100%, both of which significantly enhance the infectivity of new coronavirus variant strain.

In the research of the new crown variant strain, the applicant finds that the B.1.429 variant strain has stronger immunogenicity although the advantage of the B.1.429 variant strain in epidemic situation is gradually lost. Compared with other types of new crown variant strains, the vaccine can stimulate a broad-spectrum neutralizing antibody reaction when used for independent immunization or used as heterotypic booster immunization together with a marketed adenovirus vector new crown wild strain vaccine, has better neutralizing capacity on the new crown wild strain and mutant strains such as B.1.1.7, B.1.351, P.1, B.1.617.1, B.1.617.2 and the like, and the neutralizing capacity on a specific variant strain can approach or exceed the immunogenicity of a vaccine prepared from the specific variant strain. Has certain application potential under the background of continuous epidemic situation of the new crown variant strain.

Disclosure of Invention

Based on the above objects, the present invention provides a spike protein mutant of a novel coronavirus B.1.429 variant, wherein the amino acid sequence of the spike protein mutant is derived from the spike protein of the novel coronavirus B.1.429 variant, and is shown in SEQ ID NO. 1.

Secondly, the invention provides a polynucleotide for coding the spike protein mutant of the novel coronavirus B.1.429 variant, wherein the sequence of the polynucleotide is shown as SEQ ID NO. 2.

Thirdly, the invention provides a human type 5 replication-defective adenovirus which contains the combined deletion of the recombinant E1 and E3 of the polynucleotide. The polynucleotide takes replication-defective human adenovirus 5 with combined deletion of E1 and E3 as a vector, HEK293 cells integrating adenovirus E1 genes as a packaging cell line, and the novel coronavirus of the recombinant adenovirus vector is obtained by packaging.

Fourthly, the invention provides the application of the human type 5 replication-defective adenovirus with combined deletion of the recombinant E1 and the recombinant E3 in preparing a vaccine for preventing novel coronavirus pneumonia.

In a preferred embodiment, the human replication defective adenovirus type 5 deleted in combination with the recombinant E1, E3 is prepared as an injection, nasal drops, spray or inhalant.

In a more preferred embodiment, the human type 5 replication-defective adenovirus deleted in combination with the recombinant E1, E3 is prepared as an intramuscular injection.

Finally, the invention provides a preparation method of a human 5-type replication-defective adenovirus with combined deletion of recombinant E1 and E3 for expressing the spike protein mutant of the novel coronavirus B.1.429 variant strain, wherein the method comprises the following steps:

(1) constructing a shuttle plasmid vector containing the polynucleotide for coding the spike protein mutant of the novel coronavirus B.1.429 variant strain;

(2) co-transforming the shuttle plasmid vector obtained in the step (1) and the skeleton plasmid into a host cell, and packaging the recombinant human 5-type replication-defective adenovirus with combined deletion of E1 and E3;

(3) culturing the human type 5 replication-deficient adenovirus with combined deletion of the recombinant E1 and E3 in the step (2).

In a preferred embodiment, the shuttle plasmid vector of step (1) is pDC 316.

In another preferred embodiment, the backbone plasmid of step (2) is pBHGlox _ E1, 3 Cre.

In yet another preferred embodiment, the host cell of step (2) is a HEK293 cell.

The recombinant adenovirus capable of expressing the antigen protein of the novel coronavirus B.1.429 variant provided by the invention is used as a novel coronavirus vaccine, and has good immunogenicity after a mouse is immunized. Compared with the 5-type adenovirus vector new crown wild strain vaccine (Ad 5-nCoV), after 4 weeks of single-dose immunization, the combined antibody and the neutralizing antibody against the wild strain are basically equivalent, and the combined antibody and the neutralizing antibody against the new crown B.1.351 and B.1.617.2 variant strains are obviously enhanced. In the combined immunization study, Ad5-nCoV was used for priming, and after 4 weeks, boost was performed with b.1.429 variant vaccine, and after 2 weeks of boost, neutralizing antibodies against the wild strain of neocorona were enhanced by about 94.93-fold, neutralizing antibodies against the euvirus of the b.1.351 variant of neocorona were enhanced by about 116.62-fold, and neutralizing antibodies against the virus of the b.1.617.2 variant of neocorona were enhanced by about 77.13-fold. The Ad5-nCoV-B.1.429 variant vaccine is used in combination with the Ad5-nCoV wild vaccine for heterotypic boosting, compared with Ad5-nCoV homotypic boosting, the combined antibody, the neutralizing antibody and the pseudovirus neutralizing antibody aiming at the new crown wild strain, B.1.617.2 and B.1.351 variants are all obviously enhanced, the pseudovirus neutralizing antibody aiming at the B.1.1.7, P.1 and B.1.617.1 variants is also obviously enhanced, and the level of the vaccine which is the same with that prepared by the spike protein of the corresponding variant can be reached or exceeded. The result shows that the scheme of the invention can be used for single immunization, and can also be used together with Ad5-nCoV to induce stronger and more broad-spectrum neutralizing antibody reaction, thereby providing important vaccine candidate strain reference for coping with new crown epidemic situation.

Drawings

FIG. 1 is a comparison of GC content distribution before and after optimization of the nucleotide sequence of the novel variant strain of crown B.1.429.

FIG. 2 is a comparison of DNA secondary structure information before and after optimization of the nucleotide sequence of the new crown B.1.429 variant.

FIG. 3 is a comparison of the nucleotide sequence of the new crown B.1.429 variant with the optimized pre-and post-codon distribution.

FIG. 4 plasmid pDC316-nCoV-B.1.429 map, fragment nCoV-B.1.429-S was obtained by cloning into pDC316 shuttle plasmid after preparation.

FIG. 5 is electrophoresis diagram of expression level of S protein of vaccine of recombinant coronavirus B.1.429 variant strain. Wherein, the 'NC' group and the 'Ad 5-Null' group are respectively a cell control group and an Ad5 empty vector control group, the Ad5-nCoV is a control group of a 5-type adenovirus vector recombinant novel coronavirus wild strain vaccine, and the Ad5-nCoV-B.1.429 is an experimental group in the scheme of the application.

FIG. 6 Ad5-nCoV-B.1.429 induced the production of bound and neutralizing antibody levels. A. C, E is the level of binding antibody against the wild strain of neotame and B.1.351, B.1.617.2 respectively; B. d, F neutralizing antibody levels against the wild strain of neotame and B.1.351, B.1.617.2, respectively.

FIG. 7 comparison of neutralizing antibody levels after Ad5-nCoV priming and boosting with Ad 5-nCoV-B.1.429. A to C were reacted with neutralizing antibodies against the wild strain, B.1.351 and B.1.617.2 mutant strains before and after 2 weeks of booster immunization, respectively.

FIG. 8 binding antibody, neutralizing antibody levels after 4 weeks of heteroboost using Ad5-nCoV and Ad5-nCoV-B.1.429, in contrast to Ad5-nCoV homoboost and other variant vaccine heteroboost. A-B are respectively the level of the binding antibody and the neutralizing antibody of the wild strain, C-D are respectively the level of the binding antibody and the neutralizing antibody of the B.1.351 variant strain, and E-F are respectively the level of the binding antibody and the neutralizing antibody of the B.1.617.2 variant strain.

FIG. 9 pseudovirus neutralizing antibody levels after 4 weeks of heteroboost using Ad5-nCoV and Ad5-nCoV-B.1.429, in contrast to Ad5-nCoV homoboost and other variant vaccine heteroboost. A to F are the levels of pseudovirus neutralizing antibodies against wild strains, B.1.351, B.1.617.2, B.1.1.7, P.1 and B.1.617.1 variant strains, respectively.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

The starting plasmids, enzymes and reagents used in the following examples were all commercially available from commercial companies unless otherwise specified.

Example 1: preparation of recombinant novel coronavirus B.1.429 variant vaccine using human replication-defective adenovirus type 5 as vector

1.1 screening and design of antigen protein of new crown B.1.429 variant

We selected the sample collected from California, USA (GISAID Access ID: EPI _ ISL _ 1032411) in 1 month 2021 as a template to obtain the protein sequence of variant Spike (Spike, S). On the basis, the protein structure is mutated on the premise of ensuring the stability and the integrity of the protein and improving the expression level. First, the original signal peptide was replaced with tissue plasminogen activator signal peptide (tPA), while lysine at position 986 was replaced with proline, and valine at position 987 was replaced with proline, to improve S protein stability and expression level. In addition, arginine at 682, 683 and 685 positions is deleted, so that the cleavage site of furin in a protein sequence is knocked out, S protein is prevented from being hydrolyzed into two peptide fragments of S1 and S2, and the integrity of the antigen after expression is ensured. The corresponding optimized design mutation is respectively positioned at the N end and the C end of the S1 region of the protein and the S2 region, and is positioned outside the receptor binding region, so that the immunogenicity of the S protein can be effectively maintained. The sequence of the optimally designed variant antigen protein is shown in SEQ ID NO: 1.

1.2 optimization and synthesis of antigen protein gene of new crown B.1.429 variant

According to the designed antigen protein sequence of the variant strain after screening, the rare codon in the spike protein gene is changed into the optimal codon of the mammalian cell according to the codon preference, and the rare codon used for cloning is removedEcoRI andHinand the dii enzyme cutting site improves the content of GC basic groups, reduces the number of AU enrichment areas in mRNA and further stabilizes a nucleic acid structure, and the result is shown in figure 1.

The optimization process enhances nucleic acid stability by increasing GC content. The length of the original sequence of the spike protein is 3822bp, the base A is 1124, accounting for 29.41%, the C is 724, accounting for 18.94%, the G is 703, accounting for 18.39%, the T is 1271, accounting for 33.25% (A in figure 1), after the design and the sequence optimization are carried out according to the embodiment 1.1, the length is 3846bp, the A is 903, accounting for 23.48%, the C is 1195, accounting for 31.07%, the G is 1012, accounting for 26.31%, the T is 736, accounting for 19.14% (B in figure 1), and the GC base content is increased from 37.33% to 57.38%. Because AT pairing forms 2 hydrogen bonds and GC pairing forms 3 hydrogen bonds, the improvement of the GC base content obviously improves the DNA double-strand pairing and the stability of the mRNA secondary structure. In addition, the distribution of GC content of the optimized gene is more uniform, and a high GC content region and a high AT content region are obviously reduced. Thus, optimization significantly improved spike protein nucleic acid stability.

The optimization process enhances nucleic acid stability by improving secondary structure. The DNA-fitted secondary structure before optimization is shown in FIG. 2A, and the DNA-fitted secondary structure after optimization is shown in FIG. 2B. The gene optimization process further reduces the number of repetitive sequences and hairpin structures in DNA molecules, and the free energy of nucleic acid molecules is obviously reduced. Therefore, the optimization obviously improves the secondary structure of the nucleic acid molecule, and the stability is obviously enhanced.

The optimization process increases gene expression efficiency by reducing rare codon usage frequency. Because the optimized pre-sequence contains more mammalian rare codons, the high-efficiency expression in mammalian cells is difficult. The analysis results of the rare codons of the nucleic acid sequences before and after optimization are shown in A in figure 3 and B in figure 3, and the results show that the content of the rare codons of the spike protein nucleic acid sequence is obviously reduced, and the use frequency of the high-frequency codons is obviously improved. Increasing the content of high-frequency codons in mammals can improve the utilization efficiency of corresponding tRNA in mammalian cells, thereby improving the utilization efficiency of raw materials in protein synthesis and improving the expression level of corresponding proteins in the mammalian cells.

Secondly, a Kozak sequence (GCCGCCACC) was added before the start codon to increase the level of antigen protein expression. The gene sequence of the antigen protein of the variant strain after synthesis or preparation of the optimized design is shown in SEQ ID NO. 2.

1.3 recombinant virus vaccine packaging and in vitro expression identification

During gene synthesis, the synthesized product was cloned into pDC316 vector (Microbix Biosystems Inc., Canada), and the resulting plasmid (pDC 316-nCoV-B.1.429) map was shown in FIG. 4. The pDC316-nCoV-B.1.429 plasmid was co-transfected with the adenovirus backbone plasmid pBHGlox _ E1, 3Cre (Microbix Biosystems Inc., Canada) into HEK293 cells, maintained in culture in DMEM medium containing 5% FBS until cytopathic effect. In the process of maintaining culture, the pDC316 vector contains partial segments of adenovirus left-side inverted repeat sequences, packaging signal sequences and antigen genes, and is spliced with virus backbone sequences by virtue of Cre/Loxp site-specific recombination to form a virus complete genome, and synthesis and assembly of progeny viruses are started. With the continuous synthesis of progeny virus, cytopathic effect gradually worsens, after more than 90% of cells are completely affected and fall off from the bottom of the dish, 1000g of the cells are centrifuged for 10 minutes to collect diseased cells, a proper amount of PBS is added, and after repeated freeze thawing is carried out for 3 times at minus 80 ℃/37 ℃, the supernatant is taken to collect recombinant adenovirus, and then the recombinant adenovirus is frozen and stored at minus 80 ℃. The virus seeds are used for 3 successive generations in HEK293 cells, and third generation virus is collected and sequenced for identification.

The virus titer after verification is measured, HEK293 cells are infected by MOI =1, and the cells are collected after 24 hours for Western Blot identification, and the results are shown in FIG. 5, wherein an "NC" group and an "Ad 5-Null" group are respectively a cell control and an Ad5 empty vector control, Ad5-nCoV (CN 111218459A) is a novel coronavirus wild strain vaccine control, and Ad5-nCoV-B.1.429 is the S protein expression condition after HEK293 cells are infected by a variant vaccine of B.1.429 strain in the scheme of the application. A comparison shows that the expression level of the S protein is similar to that of Ad5-nCoV after Ad5-nCoV-B.1.429 variant strains infect cells. In addition, since the variant vaccine S protein has a furin site knocked out, the S protein is in a full-length form, while the S protein of Ad5-nCoV is partially hydrolyzed to generate an S1 band.

Example 2: evaluation of immune response of novel crown B.1.429 variant vaccine

Using an intramuscular injection, 100. mu.l of a solution containing 5X 10 of the drug was injected through the medial thigh⁸VP vaccine samples, mice bled 4 weeks after immunization, and sera separated for detection of binding, neutralizing, and pseudovirus neutralizing antibodies, respectively.

2.1 the new crown B.1.429 variant vaccine induced a higher level of binding antibody response.

Since the B.1.351 and B.1.617.2 variants were more resistant to serum from Xinguan convalescent individuals, we focused on the antibody responses of the vaccine against the Xinguan wild type (Genebank number: NC-045512.2) and the two variants. The serum was tested for specific IgG antibody titers by ELISA using the three novel coronavirus S proteins described above as antigens, as shown in A of FIG. 6, C of FIG. 6, and E of FIG. 6 (. beta., P < 0.05;. beta., P < 0.01;. beta., P < 0.001).

After 4 weeks of intramuscular injection of adenovirus vector recombinant vaccines, the antibody titers approach peak levels, so we used 4 weeks after immunization as the primary point of humoral immunity evaluation. The b.1.429 variant vaccine elicited an equal or slightly higher level of binding antibody response to wild-type S protein after immunization compared to Ad5-nCoV (a of fig. 6), whereas the binding antibody response to b.1.351 and b.1.617.2 variant S proteins was significantly higher than that of the Ad5-nCoV group (t-test, P < 0.001), reaching the latter 1.54-fold (C of fig. 6) and 1.99-fold (E of fig. 6), respectively. In addition, it is noted that the geometric mean of the antibody against b.1.351 spike protein in serum after the b.1.429 variant vaccine alone was 262613, while the geometric mean of the vaccine against b.1.351 spike protein in the variant vaccine prepared by using b.1.351 spike protein was 292046, which was not significantly different (C in fig. 6). Similarly, the geometric mean of the antibody against the spike protein of B.1.617.2 in serum after immunization of the B.1.429 variant vaccine alone was 398148, while the geometric mean of the vaccine against the spike protein of B.1.617.2 in the variant vaccine prepared was 336390, which was slightly higher than the former, and was not significantly different (E in FIG. 6).

In summary, the b.1.429 vaccine elicited significantly higher binding antibody responses in mice to wild-strain, b.1.351 and b.1.617.2 variant spike proteins than the neo-crown wild-strain vaccine, and the binding antibody response to the variant spike protein was comparable to that of vaccines prepared with the corresponding variant spike protein. The results show that the B.1.429 variant vaccine has better broad-spectrum neutralizing antibody response when singly immunized.

2.2 the novel variant vaccine of crown B.1.429 induces a higher level of neutralizing antibody response

The titer of neutralizing antibodies against the true virus was measured by microplate assay using wild strain virus, variant virus of new crown B.1.351, and variant virus of new crown B.1.617.2, and the results were shown in B in FIG. 6, D in FIG. 6, and F in FIG. 6 (P < 0.05;, P < 0.01;, P < 0.001).

Neutralizing antibodies are essentially identical to the bound antibody conclusion. After 4 weeks of immunization with the Ad5-nCoV wild strain vaccine and the Ad5-nCoV-b.1.429 variant vaccine, the geometric mean of neutralizing antibodies against the new crown wild strain in mouse serum was 92, and there was no significant difference between the two (B in fig. 6); while the neutralizing antibody responses against the B.1.351 and B.1.617.2 variants were significantly higher than those of Ad5-nCoV group (t-test, P < 0.001), reaching 4.46 times (D in FIG. 6) and 4.81 times (F in FIG. 6), respectively, of the latter.

It is also worth mentioning that the geometric mean of the neutralizing antibodies against b.1.351 in serum after immunization of the b.1.429 variant vaccine alone was 97, whereas the geometric mean of the neutralizing antibodies against b.1.351 in the variant vaccine prepared using the b.1.351 spike protein after immunization was 171, which is about 1.76 times that of the former, but there was no significant difference between the two (D in fig. 6). The geometric mean of the neutralizing antibodies against b.1.617.2 from serum after immunization alone for the b.1.429 variant vaccine was 118, whereas the geometric mean of the neutralizing antibodies from the variant vaccine prepared using the spike protein of b.1.617.2 after immunization was 182, which was about 1.54 times that of the former, and there was no significant difference between them (F in fig. 6).

In conclusion, the neutralizing antibody response of the B.1.429 variant vaccine in mice against wild strains, B.1.351 and B.1.617.2 variants is significantly higher than that of the Xinguan wild strain vaccine, and the neutralizing antibody response against the variants is close to the level of the vaccine prepared from the spike proteins of the corresponding variants. The results also show that the B.1.429 variant vaccine has a good broad-spectrum neutralizing antibody response when immunized alone.

2.3 the combination of the vaccine of the new crown B.1.429 variant strain and Ad5-nCoV for strengthening immunity, and the level of neutralizing antibody is obviously improved

We further evaluated that 4 weeks after priming with Ad5-nCoV, boosting with the Ad5-nCoV-B.1.429 variant vaccine (FIG. 7). The results show that the neutralizing antibody response is significantly enhanced after 2 weeks of booster immunization with the variant vaccine. The geometric mean value of the neutralizing antibody against the wild strain is enhanced by about 94.93 times (A, t test in figure 7, P < 0.001), the mean value of the neutralizing antibody against the B.1.351 variant is enhanced by about 116.62 times (B, t test in figure 7, P < 0.001), and the mean value of the neutralizing antibody against the B.1.617.2 variant is enhanced by 77.13 times (C, t test in figure 7, P < 0.001). The result shows that the broad-spectrum neutralizing antibody level aiming at wild type, Beta type and Delta type new corona viruses can be effectively improved by using the Ad5-nCoV-B.1.429 variant vaccine in combination with the currently marketed Ad5-nCoV wild vaccine strain, and the broad-spectrum neutralizing antibody has certain application potential.

2.4 after the new crown B.1.429 variant vaccine and Ad5-nCoV heterotype boosting, the antibody level is obviously higher than that of homotype boosting.

To further validate the superiority of the B.1.429 variant vaccine in combination with the wild-type vaccine, we compared the differences in antibody levels after Homoboost (Ad 5-nCoV + Ad 5-nCoV) and Homoboost (Ad 5-nCoV + Ad 5-B.1.429/B.1.351/B.1.617.2) immunization, 40 mice were immunized with Ad5-nCoV, and after 4 weeks 10 mice were immunized with Ad5-nCoV, Ad5-nCoV-B.1.429, Ad5-nCoV-B.1.351, Ad5-nCoV-B.1.617.2, respectively.

After 4 weeks of immunization, the levels of bound and neutralizing antibodies are shown in FIG. 8. Using B.1.429 variant vaccine to carry out heterotypic boosting, the combined antibody and neutralizing antibody aiming at the new crown wild strain are respectively enhanced by 3.63 times (A of figure 8, t test, P is less than 0.001) and 2.64 times (B of figure 8, t test, P is less than 0.01) compared with homotypic boosting; the b.1.429 variant vaccine was used for heterotypic boost, with 3.75-fold (C, t test in fig. 8, P < 0.001) and 3.12-fold (D, t test in fig. 8, P > 0.05) enhancement of the binding and neutralizing antibodies against the new crown b.1.351 variant over the homotypic boost, respectively. It is noted that the antibody level was also higher than the group of heterobooster immunizations with Ad5-nCoV-b.1.351, the geometric mean of the bound antibody was about 2.02 fold the latter (C, t test, P <0.05 in fig. 8), and the neutralizing antibody was not significantly different, but the geometric mean was about 1.85 fold the latter (D, t test, P >0.05 in fig. 8).

The b.1.429 variant vaccine was used for the hetero-boost, and the binding and neutralizing antibodies against the neo-corona b.1.617.2 variant were 3.77-fold (E, t test, P <0.001 in fig. 8) and 2.63-fold (F, t test, P <0.05 in fig. 8), respectively, higher than the homo-boost. This antibody level was close to that of the experimental group boosted with Ad5-nCoV-B.1.1.617.2, with the geometric mean of bound antibody being about 1.46 times that of the former (E, t test, P >0.05 in FIG. 8) and the geometric mean of neutralizing antibody being about 1.81 times that of the former (F, t test, P >0.05 in FIG. 8), but there was no significant difference between bound and neutralized antibodies.

As described above, the b.1.429 variant vaccine was used for the heteroplastic boost, the neutralizing antibody responses against the wild-type strain, b.1.351 and b.1.617.2 variants were significantly higher in the mice than in the homoplastic boost group, the binding and neutralizing antibody responses against the b.1.351 variant vaccine were higher than in the heteroplastic boost group using the b.1.351 variant vaccine, and the binding and neutralizing antibody responses against the b.1.617.2 variant vaccine were comparable to the heteroplastic boost group using the b.1.617.2 variant vaccine. The result shows that the B.1.429 variant vaccine can be used for heterotypic boosting immunization with a 5-type adenovirus vector new crown wild vaccine, and has better broad-spectrum neutralization effects on the new crown wild vaccine as well as B.1.351 and B.1.61.2 variants.

2.5 the broad-spectrum neutralizing activity of the new crown polytype virus strain can be stimulated by the new crown B.1.429 variant vaccine and Ad5-nCoV heterotype boosting immunity

We have synthesized wild type and B.1.1.7, B.1.351, P.1, B.1.617.1 and B.1.617.2S protein gene sequences, respectively, and embedded the sequences into pCAGGS plasmid for intracellular overexpression of the novel crown S protein. The pCAGGS plasmid and the pNL4-3. Luc-R-E-skeleton plasmid are co-transfected into a HEK293 cell line, and culture supernatants are collected after 48 hours and 72 hours to prepare the HIV skeleton new crown variant pseudovirus. The pseudovirus was used to neutralize mouse serum, followed by infection with an ACE2 stable transgenic cell line (HEK 293-ACE 2) to detect firefly luciferase expression levels for quantitative detection of mouse serum pseudovirus neutralizing antibodies after 4 weeks of booster immunization, and the results are shown in FIG. 9.

After heteroboosting Ad5-nCoV-B.1.429, higher levels of pseudovirus neutralizing antibody responses were elicited in mice for the new crown wild-type strains (A, t test, P <0.05, FIG. 9), and B.1.351 (B, t test, P <0.01, B.1.617.2 (C, t test, P <0.05, FIG. 9), B.1.1.7 (D, t test, P <0.01, FIG. 9), P.1 (E, t test, P <0.01, B.1.617.1 (F, t test, P <0.01, FIG. 9) variants as compared to Ad5-nCoV homoboosting.

Furthermore, adenovirus vector recombinant variant vaccines Ad5-nCoV-B.1.351, Ad5-nCoV-B.1.617.2, Ad 5-nCoV-B.1.1.7, Ad 5-nCoV-P.1 and Ad 5-nCoV-B.1.617.1 were prepared from the spike proteins of the B.1.351, B.1.617.2, B.1.1 variant strains, and were used in combination with Ad5-nCoV to carry out heterotypic booster immunization and to detect neutralizing antibodies against the corresponding variant pseudoviruses, respectively (corresponding to the second column data in FIGS. 6B-6F). The results show that the heteroboost immunization with Ad5-nCoV-B.1.429 elicited a slightly higher or substantially equivalent level of pseudovirus neutralizing antibody responses against the variant strain as the corresponding variant strain vaccine, with no significant difference in all groups. The results further suggest that the b.1.429 variant vaccine can be used for heterotypic boosting immunization with a 5-type adenovirus vector new crown wild strain vaccine, and can induce a stronger and broader-spectrum neutralizing antibody reaction against wild strains or multiple variant strains.

Sequence listing

<110> military medical research institute of military science institute of people's liberation force of China

<120> adenovirus vector recombinant new coronavirus B.1.429 variant vaccine and application thereof

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Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

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Ala Val Phe Val Ser Asn Ser Ile Gln Cys Val Asn Leu Thr Thr Arg

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Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr

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Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp

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Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His

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85 90 95

Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile

100 105 110

Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu

115 120 125

Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe

130 135 140

Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn

145 150 155 160

Lys Ser Cys Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn

165 170 175

Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly

180 185 190

Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile

195 200 205

Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val

210 215 220

Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu

225 230 235 240

Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His

245 250 255

Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly

260 265 270

Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu

275 280 285

Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu

290 295 300

Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu

305 310 315 320

Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser

325 330 335

Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val

340 345 350

Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg

355 360 365

Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser

370 375 380

Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp

385 390 395 400

Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp

405 410 415

Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr

420 425 430

Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn

435 440 445

Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr

450 455 460

Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser

465 470 475 480

Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly

485 490 495

Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn

500 505 510

Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu

515 520 525

Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu

530 535 540

Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr

545 550 555 560

Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe

565 570 575

Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr

580 585 590

Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val

595 600 605

Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln

610 615 620

Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu

625 630 635 640

Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr

645 650 655

Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu

660 665 670

Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln

675 680 685

Thr Asn Ser Pro Ala Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr

690 695 700

Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile

705 710 715 720

Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro

725 730 735

Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly

740 745 750

Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys

755 760 765

Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys

770 775 780

Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro

785 790 795 800

Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp

805 810 815

Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn

820 825 830

Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys

835 840 845

Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn

850 855 860

Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln

865 870 875 880

Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe

885 890 895

Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr

900 905 910

Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln

915 920 925

Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp

930 935 940

Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val

945 950 955 960

Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser

965 970 975

Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu

980 985 990

Asp Pro Pro Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg

995 1000 1005

Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala

1010 1015 1020

Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys

1025 1030 1035 1040

Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His

1045 1050 1055

Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His

1060 1065 1070

Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala

1075 1080 1085

Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val

1090 1095 1100

Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro

1105 1110 1115 1120

Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1125 1130 1135

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu

1140 1145 1150

Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr

1155 1160 1165

Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val

1170 1175 1180

Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn

1185 1190 1195 1200

Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln

1205 1210 1215

Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu

1220 1225 1230

Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys

1235 1240 1245

Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe

1250 1255 1260

Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr

1265 1270 1275 1280

Thr

<210> 2

<211> 3846

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggacgcca tgaagcgggg cctctgctgt gttctgctgc tctgcggcgc cgtgttcgtg 60

agtaactcga tccagtgcgt gaacctgacc accagaacac agctgcctcc agcctacacc 120

aacagcttca ccagaggcgt gtactacccc gacaaggtgt tcagatccag cgtgctgcac 180

tctacccagg acctgttcct gcctttcttc agcaacgtga cctggttcca cgccatccac 240

gtgagcggca ccaatggcac caagagattc gacaaccccg tgctgccctt caacgacggg 300

gtgtactttg ccagcaccga gaagtccaac atcatcagag gctggatctt cggcaccaca 360

ctggacagca agacccagag cctgctgatc gtgaacaacg ccaccaacgt ggtcatcaaa 420

gtgtgcgagt tccagttctg caacgacccc ttcctgggcg tgtactacca caagaacaac 480

aagagctgca tggaaagcga gttccgggtg tacagcagcg ccaacaactg caccttcgag 540

tacgtgtccc agcctttcct gatggacctg gaaggcaagc agggcaactt caagaacctg 600

cgcgagttcg tgttcaagaa catcgacggc tacttcaaga tctacagcaa gcacacccct 660

atcaacctcg tgcgggatct gcctcagggc ttctctgctc tggaacccct ggtggatctg 720

cccatcggca tcaacatcac ccggtttcag acactgctgg ccctgcacag aagctacctg 780

acacctggcg atagcagctc tggatggaca gctggcgccg ctgcctacta tgtgggatac 840

ctgcagcctc ggaccttcct gctgaagtac aacgagaacg gcaccatcac cgacgccgtg 900

gattgtgctc tggatcctct gagcgagaca aagtgcaccc tgaagtcctt caccgtggaa 960

aagggcatct accagaccag caacttccgg gtgcagccca ccgaatccat cgtgcggttc 1020

cccaatatca ccaatctgtg ccccttcggc gaggtgttca atgccaccag attcgcctct 1080

gtgtacgcct ggaaccggaa gcggatcagc aattgcgtgg ccgactactc cgtgctgtac 1140

aactccgcca gcttcagcac cttcaagtgc tacggcgtgt cccctaccaa gctgaacgac 1200

ctgtgcttca caaacgtgta cgccgacagc ttcgtgatcc ggggagatga agtgcggcag 1260

attgcccctg gacagacagg caagatcgcc gactacaact acaagctgcc cgacgacttc 1320

accggctgtg tgattgcctg gaacagcaac aacctggact ccaaagtcgg cggcaactac 1380

aattacagat acaggctgtt taggaagtcc aacctgaagc cctttgagcg ggatattagc 1440

accgagatct accaggccgg gagcacccct tgtaacggcg tcgaggggtt taactgctac 1500

tttcctctgc agagctacgg gttccagccc accaacgggg tcgggtacca gccataccgg 1560

gtggtggtgc tgagcttcga gctgctgcac gccccagcca ccgtctgcgg ccccaagaag 1620

tccactaacc tggtgaagaa caagtgcgtg aacttcaact tcaacggcct gacagggaca 1680

ggcgtgctga cagagtccaa caagaagttc ctccccttcc agcagtttgg gcgggacatt 1740

gccgacacaa ccgatgccgt gcgggaccca cagaccctgg agatcctgga catcacaccc 1800

tgcagcttcg gcggggtgag cgtgattaca cccggcacaa acacctccaa ccaggtggcc 1860

gtgctgtacc agggcgtgaa ctgtacagag gtgccagtgg ccattcacgc cgatcagctg 1920

acccctactt ggcgggtgta ctccacaggc agcaatgtgt ttcagaccag agccggctgt 1980

ctgatcggag ccgagcacgt gaacaatagc tacgagtgcg acatccccat cggcgctggc 2040

atctgcgcct cttaccagac acagaccaat agccctgcca gcgtggccag ccagagcatc 2100

attgcctaca caatgtctct gggcgccgag aactctgtgg cctactccaa caactctatc 2160

gctatcccca ccaacttcac catcagcgtg accacagaga tcctgcctgt gtccatgacc 2220

aagaccagcg tggactgcac catgtacatc tgcggcgatt ccaccgagtg ctccaacctg 2280

ctgctgcagt acggcagctt ctgcacccag ctgaatagag ccctgacagg gatcgccgtg 2340

gaacaggaca agaacaccca agaggtgttc gcccaagtga agcagatcta caagacccct 2400

cctatcaagg acttcggcgg cttcaatttc agccagattc tgcccgatcc tagcaagccc 2460

agcaagcgga gcttcatcga ggacctgctg ttcaacaaag tgacactggc cgacgccggc 2520

ttcatcaagc agtatggcga ttgtctgggc gacattgccg ccagggatct gatttgcgcc 2580

cagaagttta acggactgac agtgctgcct cctctgctga ccgatgagat gatcgcccag 2640

tacacatctg ccctgctggc cggcacaatc acaagcggct ggacatttgg agctggcgct 2700

gccctgcaga tcccctttgc tatgcagatg gcctaccggt tcaacggcat cggagtgacc 2760

cagaatgtgc tgtacgagaa ccagaagctg atcgccaacc agttcaacag cgccatcggc 2820

aagatccagg acagcctgag cagcacagca agcgccctgg gaaagctgca ggacgtggtc 2880

aaccagaatg cccaggcact gaacaccctg gtcaagcagc tgtctagcaa cttcggcgcc 2940

atctctagcg tgctgaacga tatcctgagc agactggacc ctcctgaagc cgaggtgcag 3000

atcgacagac tgatcaccgg aaggctgcag tccctgcaga cctacgttac ccagcagctg 3060

atcagagccg ccgagattag agcctctgcc aatctggccg ccaccaagat gtctgagtgt 3120

gtgctgggcc agagcaagag agtggacttt tgcggcaagg gctaccacct gatgagcttc 3180

cctcagtctg ctcctcacgg cgtggtgttt ctgcacgtga catacgtgcc cgctcaagag 3240

aagaatttca ccaccgctcc agccatctgc cacgacggca aagcccactt tcctagagaa 3300

ggcgtgttcg tgtccaacgg cacccattgg ttcgtgaccc agcggaactt ctacgagccc 3360

cagatcatca ccacagacaa caccttcgtg tccggcaact gcgacgtcgt gatcggcatt 3420

gtgaacaata ccgtgtacga ccctctgcag cccgagctgg acagcttcaa agaggaactg 3480

gataagtact ttaagaacca cacaagcccc gacgtggacc tgggcgatat cagcggaatc 3540

aatgcctccg tcgtgaacat ccagaaagag atcgaccggc tgaacgaggt ggccaagaat 3600

ctgaacgaga gcctgatcga cctgcaagaa ctggggaagt acgagcagta catcaagtgg 3660

ccttggtaca tctggctggg ctttatcgcc ggactgattg ccatcgtgat ggtcacaatc 3720

atgctgtgtt gcatgaccag ctgctgtagc tgcctgaagg gctgttgtag ctgtggctcc 3780

tgctgcaagt tcgacgagga cgattctgag cccgtgctga aaggcgtgaa gctgcactac 3840

acctga 3846

Claims

1. A polynucleotide molecule for coding a spike protein mutant of a novel coronavirus B.1.429 variant strain, wherein the sequence of the polynucleotide molecule is shown as SEQ ID NO. 2.

2. A human type 5 replication-defective adenovirus containing the polynucleotide molecule of claim 1 and deleted in combination of recombinant E1 and E3, wherein the human type 5 replication-defective adenovirus deleted in combination of recombinant E1 and E3 expresses a spike protein mutant of the variant coronavirus B.1.429 encoded by the polynucleotide molecule of claim 1.

3. Use of the human type 5 replication-defective adenovirus deleted in combination of the recombinant E1 and E3 of claim 2 in the preparation of a vaccine for preventing novel coronavirus pneumonia.

4. The use according to claim 3, wherein the recombinant E1, E3 combined deleted human type 5 replication defective adenovirus is prepared as an injection, nasal drop, spray or inhalant.

5. The use according to claim 4, wherein the recombinant E1, E3 combined deleted human type 5 replication defective adenovirus is prepared as an intramuscular injection.

6. A method for preparing a human replication defective adenovirus type 5 with a combined deletion of recombinant E1 and E3 according to claim 2, the method comprising the steps of:

(1) constructing a shuttle plasmid vector comprising the polynucleotide molecule of claim 1;

(2) co-transforming the shuttle plasmid vector obtained in the step (1) and a skeleton plasmid into a host cell, and packaging the human type 5 replication-defective adenovirus subjected to combined deletion of recombinant E1 and E3;

(3) culturing the recombinant human 5-type replication-defective adenovirus with combined deletion of E1 and E3 obtained in the step (2).

7. The method of claim 6, wherein the shuttle plasmid vector of step (1) is pDC 316.

8. The method of claim 6, wherein the backbone plasmid of step (2) is pBHGlox _ E1, 3 Cre.

9. The method according to claim 6, wherein the host cell of step (2) is HEK293 cell.