CN117210503A

CN117210503A - Recombinant adeno-associated virus vector for expressing nanobody and application thereof

Info

Publication number: CN117210503A
Application number: CN202311480579.1A
Authority: CN
Inventors: 郝睿; 王仲亚; 张庆硕; 高红英
Original assignee: Renoviron Suzhou Biotechnology Co ltd
Current assignee: Renoviron Suzhou Biotechnology Co ltd
Priority date: 2023-11-08
Filing date: 2023-11-08
Publication date: 2023-12-12
Anticipated expiration: 2043-11-08
Also published as: CN117210503B

Abstract

The invention discloses a recombinant adeno-associated virus vector for expressing a nano antibody and application thereof. The invention obtains an optimized expression element combination for endogenously expressing the nano antibody by AAV through screening, and specifically comprises a CAG promoter, a BM40 signal peptide and an SV40 polyA tail. The AAV can be packaged by adeno-associated virus and injected into human body, so that the expression level of the AAV for delivering nanobody into human body can be raised, and the expression of nanobody in human body can be maintained at high level for a long period. The AAV vector realizes the stable and efficient expression of the nano antibody drug in vivo, and has wide application value in the fields of disease treatment, diagnosis, substance detection and the like.

Description

Recombinant adeno-associated virus vector for expressing nanobody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant adeno-associated virus vector for expressing a nano antibody and application thereof.

Background

Adeno-associated virus (AAV) is the most widely used therapeutic means in the current gene therapy project, and has the advantages of wide host range, high safety, low immunogenicity, stable expression, stable physical properties and the like. Most of the gene therapy drugs to date are directed to rare diseases, and the market range is limited, so that the AAV therapeutic range is expanded, and the AAV therapeutic range is not limited to the rare diseases any more, and the AAV therapeutic drug has a broad prospect.

AAV-based antibody therapy is a potential strategy to effectively expand its therapeutic range. For example, the National Institutes of Health (NIH) published 2022 clinical results for gene therapy of HIV with AAV viruses, in which they delivered DNA encoding neutralizing antibodies (bnAbs) via AAV viruses that were able to produce the neutralizing antibodies in vivo, thereby achieving the goal of treating HIV. While studies of Voyager Therapeutics for the treatment of Alzheimer's Disease (AD) by AAV delivery of Tau antibodies have been successful in earlier work, they have expected IND reporting in 2024. The company vector y is developing antibody VTx-002 for treating Amyotrophic Lateral Sclerosis (ALS). However, the antibodies used in the gene therapy have been usually immunoglobulin IgG or single chain antibody (scFV), and these antibodies have a large molecular weight and high immunogenicity. Nanobodies (VHHs) have become a new choice compared to traditional antibodies. The nano antibody has small molecular weight, and is easy to permeate into dense tissues and even blood brain barriers; high affinity, strong specificity, high solubility and good stability; low immunogenicity, simple structure and easy transformation. However, nanobodies currently face an important challenge in that they have a short half-life and often do not achieve the desired therapeutic effect. To solve this problem, it is critical to increase the expression level of nanobodies in vivo and maintain the antibody level in vivo with high efficiency.

Disclosure of Invention

In order to solve the problems, the invention screens and optimizes various expression elements for delivering the nanobody through AAV, including a promoter, an intron, a signal peptide and polyA, and finally obtains an optimal combination mode, and compared with other common expression element combinations, the element combination can improve the efficiency of expressing the nanobody by more than 100 times, and can maintain higher concentration of serum nanobody for a long time, thereby realizing continuous and efficient expression of the nanobody in vivo after the delivery of the nanobody through AAV.

The first object of the present invention is to provide a recombinant adeno-associated virus vector expressing nanobody, wherein the recombinant adeno-associated virus vector contains a CAG promoter, a BM40 signal peptide and an SV40 polyA tail, and the CAG promoter and the BM40 signal peptide are positioned upstream of the nanobody coding sequence, and the SV40 polyA tail is positioned downstream of the nanobody coding sequence.

Further, the recombinant adeno-associated virus vector contains an Inverted Terminal Repeat (ITR) for AAV virus packaging, and then the recombinant vector is packaged by adeno-associated virus to prepare a related product.

Furthermore, AAV expression vectors of recombinant nanobodies are used for endogenous high expression of nanobodies in patients or experimental animals.

Further, the AAV expression vector can maintain a high level of nanobody in experimental animals for a long period of time, and the nanobody concentration in vivo is substantially stable for 90 days or more after virus injection for 60 days.

It is a second object of the present invention to provide a delivery system comprising the recombinant adeno-associated viral vector described above.

Further, the delivery system is a drug delivery system, and the drug classes include, but are not limited to, nucleic acid drugs, protein drugs, and the like.

A third object of the present invention is to provide the use of the recombinant adeno-associated viral vector described above for the preparation of nanobody delivery vectors.

In the invention, the method for screening the optimal nano antibody expression element comprises the following steps:

(1) Construction of expression vector pssMMAAV-RNY-00XX

And adding a sequence for encoding nano luciferase at the C end of the nano antibody coding sequence to serve as a reporter gene for detecting the expression level. Subsequently, different promoters, introns, signal peptides were added upstream of the sequence and different polyA terminators were added downstream, giving a total of 17 combined sequences containing different expression elements, which were ligated into adeno-associated viral vectors.

(2) Extraction of expression vector pssMMAAV-RNY-00XX

The method comprises the steps of using a Tiangen endotoxin-free plasmid middling extraction kit (DP 108), operating according to a kit using instruction, taking 1 mu L of extracted plasmid, measuring the concentration by a nucleic acid tester (Nanodrop 0 ne), taking 500 ng plasmid, running gel electrophoresis, detecting and verifying, and using the extracted plasmid for AAV virus packaging and production.

(3) AAV packaging and purification

AAV293 cells were plated one day in advance, cells were transfected using a three plasmid packaging system (AdHelper, AAVDJ, vector of interest), and cell supernatants were harvested 96 hours after transfection. Cell supernatants were subjected to tangential flow filtration (Tff) and saline replacement, and finally the resulting samples were sterilized, filtered and QPCR quantified, AAV packaged and purified.

(4) U251, AAV293 and C2C12 cell infection

One day in advance, U251, AAV293 and C2C12 cells were plated into a 48-well plate, after 24 hours, the corresponding number of virus-infected cells was taken, the number of virus-infected cells=the number of cells at the time of infection (2 times the number of passaged-counted cells) xMOI (10E 5), after 72 hours of infection, the cell supernatants were taken, and the expression level of the Nano-Luc fusion protein was detected using a Nano-Glo Luciferase (Promega N1120) kit.

(5) Construction of novel expression vector pssMMAAV-RNY-00XX

According to the results of U251, AAV293 and C2C12 cell infection, three novel pssMMAAV-RNY-00XX vectors, respectively 0018, 0019 and 0020, are combined, the vector construction method is the same as that of (1), and all the vectors are subjected to sequencing verification.

(6) Novel expression vector extraction and AAV packaging

Extracting: the method comprises the steps of using a Tiangen endotoxin-free plasmid middling extraction kit (DP 108), operating according to a kit using instruction, taking 1 mu L of extracted plasmid, measuring concentration by a nucleic acid tester (Nanodrop 0 ne), taking 500 ng plasmid, running gel electrophoresis, detecting and verifying, and using the extracted plasmid for AAV virus packaging.

AAV packaging: AAV293 cells were plated one day in advance, cells were transfected using a three plasmid packaging system (AdHelper, AAV9, vector of interest), and cell supernatants were harvested 96 hours after transfection. Cell supernatants were subjected to tangential flow filtration (Tff), chromatography and saline replacement, and finally the resulting samples were sterilized, filtered and QPCR quantified, packaged and purified.

(7) AAV293 cell infection

AAV293 cells were plated into 48 well plates one day in advance, 24 hours later, the corresponding number of virus-infected cells was taken, the number of virus-infected cells = the number of cells infected (2 times the number of passaged cells) xMOI (10E 5), 72 hours later after infection, the cell supernatants were taken, and the expression level of the Nano-Luc fusion protein was detected using the Nano-Glo Luciferase (Promega N1120) kit.

(8) In vivo screening of nanobody expression elements

AAV combined with pssmmav-RNY-0001, pssmmav-RNY-00016, pssmmav-RNY-0018, pssmmav-RNY-0019 and pssmmav-RNY-0020 expression elements was selected and intravenously injected into mice, each of which was injected with 1e12 vg of the corresponding virus, and blood was withdrawn at time points of 0 day, 10 day, 15 day, 21 day, 28 day, 42 day, 61 day and 90 day, and serum was withdrawn to detect the expression level of nanobody.

A fourth object of the present invention is to provide a method for recombinantly expressing nanobodies using adeno-associated virus, comprising the steps of:

the nanobody coding sequence is linked to an adeno-associated viral vector, and the CAG promoter and BM40 signal peptide are linked upstream and the SV40 polyA tail is linked downstream of the nanobody coding sequence.

Further, an Inverted Terminal Repeat (ITR) is ligated upstream of the CAG promoter and downstream of the SV40 polyA tail, respectively.

Further, the two ITRs are identical but opposite in direction.

Further, the CAG promoter has a sequence shown in SEQ ID NO.5, the BM40 signal peptide has a sequence shown in SEQ ID NO.6, the SV40 polyA tail has a sequence shown in SEQ ID NO.7, and the inverted terminal repeat has a sequence shown in SEQ ID NO. 8.

A fifth object of the present invention is to provide a product for detecting sortilin 1 molecular level or increasing granulin precursor level, wherein the product contains recombinant adeno-associated virus, wherein the recombinant adeno-associated virus contains sortilin 1 specific nanobody, and the nanobody is connected with CAG promoter and BM40 signal peptide at the upstream and SV40 polyA tail at the downstream.

Further, the product can be a drug, such as a drug for preventing or treating neurodegenerative diseases, or can be other biological products.

Further, the nanobody specific for sortilin 1 may be at least nanobody Nb107, nb110, nb118, nb128, nb145, nb151, nb171, nb184, nb252 described in CN202310995805.3, and the sequence of CDR regions of the above nanobody is also described in the patent.

It is a sixth object of the present invention to provide the use of the recombinant adeno-associated virus described above for the preparation of a product for detecting sortilin 1 molecular level or for increasing the level of a granule protein precursor.

Further, the product for detecting sortilin 1 molecular level or increasing granulin precursor level is a drug, preferably a neurodegenerative disease drug.

The invention has the beneficial effects that:

the recombinant AAV expression element vector pssMMAAV-RNY-0020 provided by the invention has a simple construction process, can effectively promote the expression level of the nanobody in a mammal body, and helps the continuous and efficient expression of the nanobody in the body, so that the problem of low in-vivo concentration of the nanobody drug due to short half-life at present can be solved. The AAV delivery gene based on the expression element combination has the expression level higher than other preferable combinations by more than 10 times, is higher than the common expression element combination (pssMMAAV-RNY-0001) by more than 100 times, and can maintain higher nanobody level in serum for a long time.

Drawings

FIG. 1 is a map of the pssMMAAV-RNY-0020 vector.

FIG. 2 shows the result of plasmid extraction run of pssMMAAV-RNY-00XX (1-17).

FIG. 3 shows the combined expression levels of different nanobody expression elements in U251 cells.

FIG. 4 shows the combined expression levels of different nanobody expression elements in C2C12 cells.

FIG. 5 shows the combined expression levels of different nanobody expression elements in AAV293 cells.

FIG. 6 shows the residual levels of various nanobody expression elements in U251 cells.

FIG. 7 shows the residual levels of various nanobody expression elements in C2C12 cells.

FIG. 8 shows residual levels of various nanobody expression elements in AAV293 cells.

FIG. 9 is a comparison of different expression elements in U251 cells.

FIG. 10 is a comparison of different expression elements in AAV293 cells.

FIG. 11 shows a comparison of different expression elements in C2C12 cells.

FIG. 12 shows the results of plasmid extraction run of pssMMAAV-RNY-00XX (18, 19, 20).

FIG. 13 shows expression levels of novel expression element combinations in AAV293 cells.

FIG. 14 is a summary of fold increase in VHH-nanoLuc in vivo screening.

FIG. 15 shows the in vivo screening results (day 5) for the optimal nanobody expression element combination.

FIG. 16 shows the in vivo screening results (day 10) for the optimal nanobody expression element combination.

FIG. 17 shows the in vivo screening results (day 15) for the optimal nanobody expression element combination.

FIG. 18 shows the in vivo screening results (day 21) for the optimal nanobody expression element combination.

FIG. 19 shows the in vivo screening results (day 28) for the optimal nanobody expression element combination.

FIG. 20 is an in vivo expression level of the expression element combination No. 20 as a function of time.

FIG. 21 shows the expression levels of two nanobodies (Nb 171 and Nb 184) in mice (day 0, day 5, day 10, day 21) in combination with the expression element No. 20.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

The material and the method related by the invention are as follows:

the Nanoluc signal was detected using the Promega Nano-Glo Luciferase Assay System kit, cat# N1110. The operation is detected according to the specification of the kit.

Example 1

17 nanobody expression element combination vector construction

Taking the anti-Sort 1 nanobody Nb171 screened by the company as an example (recorded in China patent with the application number of CN202310995805.3, the amino acid sequences of CDR 1-CDR 3 are shown in SEQ ID NO. 1-3), and adding a sequence for encoding Nano luciferase at the C end of the nanobody coding sequence, wherein the fusion protein of the nanobody is used as a reporter gene (Nano-Luc, SEQ ID NO. 4) for detecting the expression level. On the basis, sequences such as different promoters, introns, signal peptides and the like are added at the upstream of the sequences, sequences such as different polyA terminators and the like are added at the downstream of the sequences, a total of 17 combined sequences containing different expression elements are obtained, 17 nanobody expression element combinations are synthesized by An Sheng, and the combination modes are shown in Table 1. These 17 nanobody expression element combinations were constructed by homologous recombination onto pssMMAAV plasmids (schematic see FIG. 1) retaining two inverted terminal repeats ITR for subsequent viral packaging, all obtained vectors were confirmed by sequencing. Primers suitable for homologous recombination were designed and specific sequence information is shown in Table 2. 17 nanobody expression element combinations were amplified by PCR, after which the vector was double digested with restriction enzymes NotI and SnaBI (NEB). Then, homologous recombination was performed by GeneArt ™ Gibson Assembly HiFi (Thermo Scientific A46628), and the monoclonal was picked up and sent to An Sheng for sequencing, and plasmid extraction was performed with the clones sequenced correctly. The detection and verification result of the plasmid extraction gel running electrophoresis is shown in fig. 2, wherein the left band of each figure is Marker, lanes 1-17 are recombinant plasmids pssMMAAV-RNY-0001-0017, and the extracted plasmids are used for AAV virus packaging and production.

TABLE 1 17 nanobody expression element combinations

TABLE 2 primers for PCR amplification of 17 combinations by homologous recombination

Example 2

17 nanobody expression element combined virus cell infection detection

17 nanobody expression element combination virus infects glioma cell U251: u251 cells were plated one day in advance into 48 well cell plates, 3.5E4 cells per well. At 37 ℃,5% CO ₂ The cells were incubated in the incubator for 24 hours, after which 7E9 viral load was added to each well, 3 replicates per sample. Number of infectious virus = number of cells at transfection (2 times the number of passaged cells) xMOI (10E 5). Cell supernatants were harvested 72 hours from infection and assayed for expression levels of various nanobody expression element combinations using the Nano-Glo Luciferase Assay System (Promega N1110) reagent according to the instructions. The results show that combinations No.4, 14, 15 and 16 are expressed more than the control, and the results are shown in fig. 3.

17 nanobody expression element combination virus infects mouse myoblasts C2C12: C2C12 cells were plated one day in advance into 48 well cell plates, 2E4 cells per well. At 37 ℃,5% CO ₂ The cells were incubated in the incubator for 24 hours, after which 4E9 viral load was added to each well, 3 replicates per sample. Number of infectious viruses = number of cells at transfection (number of passaged cells2 times) xMOI (10E 5). Cell supernatants were harvested 72 hours from infection and assayed for expression levels of various nanobody expression element combinations using the Nano-Glo Luciferase Assay System (Promega N1110) reagent according to the instructions. The results show that combinations No.4, 14, 15 and 16 are expressed more than the control, and the results are shown in fig. 4.

17 nanobody expression element combination virus infects human embryonic kidney cells AAV293: AAV293 cells were plated one day in advance into 48 well cell plates, 3E4 cells per well. At 37 ℃,5% CO ₂ The cells were incubated in the incubator for 24 hours, after which 6E9 viral load was added to each well, 3 replicates per sample. Number of infectious virus = number of cells at transfection (2 times the number of passaged cells) xMOI (10E 5). Cell supernatants were harvested 72 hours from infection and assayed for expression levels of various nanobody expression element combinations using the Nano-Glo Luciferase Assay System (Promega N1110) reagent according to the instructions. The results show that combinations No.4, 14, 15 and 16 are expressed more than the control, and the results are shown in fig. 5.

Example 3

Virus DNA level detection after 17 nanobody expression element combined virus infection cells

Residual DNA detection after infection of glioma cell U251 with 17 nanobody expression element combination virus: after virus infection, the infected U251 cells are obtained by digestion with pancreatin. DNA in U251 cells was extracted with a DNA extraction kit (QIAGNE 51404). The extraction method comprises the following steps: adding 200 mu L of AVE, 40 mu L of VXL, 1 mu L of DX, 20 mu L of proteinase K and 4 mu L of RNase A (100 mg/mL) into each sample, uniformly mixing, and then placing the mixture into a metal bath at 50 ℃ for 1 hour; then 265 mu L MVL is added, evenly mixed, transferred into QIAamp Mini Spin Column and centrifuged for 1 minute, the liquid receiving pipe is discarded, and a new liquid receiving pipe is replaced; centrifuging for 1 minute after adding 500 mu L of AW1 into each sample, discarding the liquid receiving pipe, and replacing the liquid receiving pipe with a new liquid receiving pipe; centrifuging for 1 minute after adding 500 mu L of AW2 into each sample, discarding the liquid receiving pipe, and replacing the liquid receiving pipe with a new liquid receiving pipe; empty centrifugation for 2 min, after which the membrane column was placed in a new 1.5 mL EP tube; the mixture was centrifuged with 50% uL sterile water for 1 min and the elution was repeated once. The reagents used above are all provided for the kit. After 100 times dilution of the DNA sample, the residual viral DNA amount is detected by Q-PCR, the Q-PCR standard is a viral plasmid enzyme-cut fragment, and a standard curve is prepared by 8 times and 4 times gradient dilution. The enzyme used for Q-PCR was SsoAdvanced Universal Probes Supermix (BIO RAD 1725281), and the probe and primer sequences are shown in Table 3:

TABLE 3Q probes and primer sequences for PCR

The results indicated that the residual viral DNA was higher for viruses 8, 9 and 10 and lower or even levels of residual DNA in the cells for viruses 4, 14, 15 and 16. The level of viral residual DNA in the cells was not correlated with the antibody expression levels in each cell, indicating that the combination of expression elements was responsible for the differences in antibody expression secretion levels. The results are shown in FIG. 6.

Residual DNA detection after infection of mouse myoblasts C2C12 with 17 nanobody expression element combined virus: after virus infection, the infected C2C12 cells are obtained by digestion with pancreatin. DNA was extracted from the C2C12 cells using the DNA extraction kit (QIAGNE Cat#: 51404). The extraction method comprises the following steps: adding 200 mu L of AVE, 40 mu L of VXL, 1 mu L of DX, 20 mu L of proteinase K and 4 mu L of RNase A (100 mg/mL) into each sample, uniformly mixing, and then placing the mixture into a metal bath at 50 ℃ for 1 hour; then 265 mu L MVL is added, evenly mixed, transferred into QIAamp Mini Spin Column and centrifuged for 1 minute, the liquid receiving pipe is discarded, and a new liquid receiving pipe is replaced; centrifuging for 1 minute after adding 500 mu L of AW1 into each sample, discarding the liquid receiving pipe, and replacing the liquid receiving pipe with a new liquid receiving pipe; centrifuging for 1 minute after adding 500 mu L of AW2 into each sample, discarding the liquid receiving pipe, and replacing the liquid receiving pipe with a new liquid receiving pipe; empty centrifugation for 2 min, after which the membrane column was placed in a new 1.5 mL EP tube; the mixture was centrifuged with 50% uL sterile water for 1 min and the elution was repeated once. The reagents used above are all provided for the kit. After 100 times dilution of the DNA sample, the residual viral DNA amount is detected by Q-PCR, the Q-PCR standard is a viral plasmid enzyme-cut fragment, and a standard curve is prepared by 8 times and 4 times gradient dilution. The enzyme used for Q-PCR was SsoAdvanced Universal Probes Supermix (BIO RAD 1725281), and the probe and primer sequences are shown in Table 4:

TABLE 4Q probes and primer sequences for PCR

The results showed that residual viral DNA was higher for numbers 8, 9 and 10, and lower than average intracellular DNA levels following infection with numbers 4, 14, 15 and 16. The level of viral residual DNA in the cells was not correlated with the antibody expression levels in each cell, indicating that the combination of expression elements was responsible for the differences in antibody expression secretion levels. The results are shown in FIG. 7.

Residual DNA detection after 17 nanobody expression element combination viruses invade human embryonic kidney cells AAV293: after virus infection, the infected AAV293 cells were obtained by digestion with pancreatin. DNA from AAV293 cells was extracted using the DNA extraction kit (QIAGNE 51404). The extraction method comprises the following steps: adding 200 mu L of AVE, 40 mu L of VXL, 1 mu L of DX, 20 mu L of proteinase K and 4 mu L of RNase A (100 mg/mL) into each sample, uniformly mixing, and then placing the mixture into a metal bath at 50 ℃ for 1 hour; then 265 mu L MVL is added, evenly mixed, transferred into QIAamp Mini Spin Column and centrifuged for 1 minute, the liquid receiving pipe is discarded, and a new liquid receiving pipe is replaced; centrifuging for 1 minute after adding 500 mu L of AW1 into each sample, discarding the liquid receiving pipe, and replacing the liquid receiving pipe with a new liquid receiving pipe; centrifuging for 1 minute after adding 500 mu L of AW2 into each sample, discarding the liquid receiving pipe, and replacing the liquid receiving pipe with a new liquid receiving pipe; empty centrifugation for 2 min, after which the membrane column was placed in a new 1.5 mL EP tube; the mixture was centrifuged with 50% uL sterile water for 1 min and the elution was repeated once. The reagents used above are all provided for the kit. After 100 times dilution of the DNA sample, the residual viral DNA amount is detected by Q-PCR, the Q-PCR standard is a viral plasmid enzyme-cut fragment, and a standard curve is prepared by 8 times and 4 times gradient dilution. The enzyme used for Q-PCR was SsoAdvanced Universal Probes Supermix (BIO RAD 1725281), and the probe and primer sequences are shown in Table 5:

TABLE 5Q probes and primer sequences for PCR

The results showed that residual viral DNA was higher for numbers 8, 9 and 10 and lower or near average levels for intracellular residual DNA following infection with numbers 4, 14, 15 and 16. The level of viral residual DNA in the cells was not correlated with the antibody expression levels in each cell, indicating that the combination of expression elements was responsible for the differences in antibody expression secretion levels. The results are shown in FIG. 8.

Example 4

Construction of element re-optimization to form 3 new nanobody expression element combination vectors

Based on the results of U251, AAV293 and C2C12 cell infection, we have arranged the effect of different selection of each class of elements (including promoters, introns, signal peptides, and polyA termination signals) on nanobody expression levels during transfection of different cells (fig. 9-11). In terms of promoter, CMV expression is best in U251 and AAV293 cells, and CAG expression is best in C2C12 cells. Intron-free, U251, AAV293 and C2C12 cells are all best. In terms of signal peptide, U251 and AAV293 are the best expressed in BM40 and C2C12 is the best expressed in HAS LP. In terms of Poly A, U251, AAV293 and C2C12 cells were all the most well expressed for SV40 Poly A. Based on the above results, three new vectors were combined, pssMMAAV-RNY-0018, pssMMAAV-RNY-0019 and pssMMAAV-RNY-0020, respectively. The specific scheme is as follows: the 3 nanobody expression element combinations were synthesized by An Sheng da corporation in the manner shown in table 6. These 3 nanobody expression element combinations were constructed onto pssMMAAV plasmids (see FIG. 1) by homologous recombination for subsequent viral packaging. Primers suitable for homologous recombination were designed and specific sequence information is shown in Table 2. The 3 nanobody expression element combinations were amplified by PCR, after which the vector was double digested with NotI and SnaBI. Then, homologous recombination was performed by GeneArt ™ Gibson Assembly HiFi (Thermo Scientific A46628), and the monoclonal was picked up and sent to An Sheng for sequencing, and plasmid extraction was performed with the clones sequenced correctly. The detection and verification result of the plasmid extraction gel running electrophoresis is shown in FIG. 12, wherein the left lanes are Marker, and lanes 18-20 are recombinant plasmid pssMMAAV-RNY-0018-0020 respectively.

Table 6 3 novel nanobody expression element combinations

Example 5

3 novel nanobody expression element combined virus-infected cells

Human embryonic kidney cell AAV293 is infected by virus using vectors No.1 and No. 16 and 3 novel nanobody expression element combinations respectively: AAV293 cells were plated one day in advance into 48 well cell plates, 3E4 cells per well. At 37 ℃,5% CO ₂ The cells were incubated in the incubator for 24 hours, after which 6E9 viral load was added to each well, 3 replicates per sample. Number of infectious virus = number of cells at transfection (2 times the number of passaged cells) xMOI (10E 5). Cell supernatants were harvested 72 hours after infection, and the results of testing the expression levels of the various nanobody expression element combinations (see FIG. 13) using Nano-Glo Luciferase Assay System (Promega N1110) reagent according to the instructions showed a significant 5-fold increase in the 16 and 20 combinations compared to No.1 and a significant 8-fold increase in the 18 and 19 combinations compared to No. 1.

Example 6

Nanobody expression element combination in vivo screening

Combinations 1, 16, 18, 19 and 20 were selected and injected intravenously with 1E12 virus per mouse tail, 7-8 week BALB/C females, 5 or 6 mice per combination. Mouse sera were taken at day 0, day 5, day 10, day 15, day 21, and day 28 and tested for expression levels in various nanobody expression element combinations by Nano-Glo Luciferase Assay System (Promega N1110) reagent according to the instructions. The results are shown in FIGS. 14, 15-19. The results showed that the combination No. 20 was most expressed in vivo at each stage of the assay, and that No. 20 was increased by 214-fold, 170-fold, 120-fold, 112-fold and 83-fold compared to No.1 at the 5-day, 10-day, 15-day, 21-day and 28-day assays, respectively. The results of in vivo experiments in mice show that the secretion expression level of the nanobody is far ahead of other element combinations under the drive of the 20 # expression element combination.

To test the stability of endogenously expressed nanobodies with AAV, we continued to culture mice injected with the highest expression level of the 20 th combination and examined the expression level of nanobodies in serum by taking blood at 42 days, 61 days and 90 days, respectively, as shown in fig. 20. It can be seen that the nanobody levels in the initial 5-10 day mouse serum increased rapidly and peaked at day 21 with a slight subsequent decrease, substantially stable at day 61 and no further decrease at day 90, indicating that serum nanobody can be maintained at a higher level for a long period of time by sustained expression of AAV endogenous, a feature consistent with the findings of NIH scientists that antibody of the IgG type was expressed with AAV.

In order to examine whether the expression element combination found by us is suitable for endogenous high expression of other nanobodies, we replaced the antibody gene Nb171 in the expression vector of the optimal combination pssMMAAV-RNY-0020 with another nanobody Nb184 (the antibody sequence is described in the Chinese patent with the application number CN202310995805.3, and the amino acid sequences of CDR 1-CDR 3 are shown in SEQ ID NO. 10-13), so that the AAV virus vector with Nb 184-nano luciferase fusion protein can be packaged. The AAV virus carrying the Nb184 nanobody gene is used for infecting a BALB/C mouse, the AAV virus 20 carrying the Nb171 nanobody with the same structure is used for parallel control, and nano luciferase activity in serum of the mouse is detected on days 5, 10 and 21, and the result is shown in figure 21. It can be seen that under the same number 20 expression element combination, the expression levels of different nanobodies in mice were very similar, which levels were much higher than other expression element combinations under equivalent conditions (see fig. 13-18). The expression element combination which is selected by us and comprises the CAG promoter, the BM40 signal peptide and the SV40 polyA tail is suitable for endogenous high expression of different nanobodies.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims

1. A recombinant adeno-associated virus vector for expressing nanobodies, which is characterized in that the recombinant adeno-associated virus vector contains a CAG promoter, a BM40 signal peptide and an SV40 polyA tail, wherein the CAG promoter and the BM40 signal peptide are positioned at the upstream of a nanobody coding sequence, and the SV40 polyA tail is positioned at the downstream of the nanobody coding sequence.

2. The recombinant adeno-associated viral vector according to claim 1, wherein the recombinant adeno-associated viral vector comprises an inverted terminal repeat.

3. A delivery system comprising the recombinant adeno-associated viral vector of claim 1 or 2.

4. Use of the recombinant adeno-associated viral vector of claim 1 or 2 for the preparation of nanobody delivery vectors.

5. A method for recombinantly expressing nanobodies using adeno-associated virus comprising the steps of: the nanobody coding sequence is linked to an adeno-associated viral vector, and the CAG promoter and BM40 signal peptide are linked upstream and the SV40 polyA tail is linked downstream of the nanobody coding sequence.

6. The method of claim 5, wherein the inverted terminal repeat is ligated upstream of the CAG promoter and downstream of the SV40 polyA tail, respectively.

7. A product for detecting sortilin 1 molecular level or increasing granulin precursor level, wherein the product comprises recombinant adeno-associated virus, wherein the recombinant adeno-associated virus comprises sortilin 1 specific nanobody, and the nanobody is connected with a CAG promoter and a BM40 signal peptide at the upstream and an SV40 polyA tail at the downstream.

8. The product of claim 7, wherein the product is a pharmaceutical, diagnostic or test article.

9. Use of a recombinant adeno-associated virus comprising a nanobody specific for sortilin 1, wherein the nanobody has a CAG promoter and a BM40 signal peptide attached upstream and an SV40 polyA tail attached downstream, for the preparation of a product for detecting sortilin 1 molecular levels or for increasing granulin precursor levels.

10. The use according to claim 9, wherein the product for detecting sortilin 1 molecular level or increasing granulin precursor level is a neurodegenerative disease drug.