CN118308390A - Intein combined carrier and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and discloses an intein combined carrier and application thereof. The intein combination vector comprises a first expression vector, a second expression vector and a third expression vector; the first expression vector comprises a promoter sequence, a target polypeptide N-terminal sequence and a first intein N-terminal sequence; the second expression vector comprises a promoter sequence, a first intein C-terminal sequence, a target polypeptide intermediate sequence and a second intein N-terminal sequence; the third expression vector comprises a promoter sequence, a second intein C-terminal sequence and a target polypeptide C-terminal sequence. The intein combined vector provided by the invention has the advantages that a plurality of insertion sites can be selected, the shearing efficiency is high, meanwhile, no cross mismatch reaction exists between the inteins, the correct splicing of target polypeptides is ensured, and the intein combined vector has important significance in the aspects of expression and production of long polypeptides, gene therapy and the like.

Description

Intein combined carrier and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an intein combined carrier and application thereof.

Background

Genetic diseases are caused by gene mutation, and about 7000 genetic diseases affect the health of people. Over 10% of patients have no effective treatment. Few drugs approved for the treatment of genetic diseases do not address the underlying genetic cause of the disease, at best only controlling or altering symptoms. Therefore, these drugs must be administered for a lifetime. Gene therapy, i.e. one-time repair or change of the affected genes of individuals, greatly improves the current situation of incurable genetic diseases and brings hopes to patients.

Adeno-associated viral (AAV) vectors are currently the most mature viral vectors in clinical trials of in vivo gene therapy. Including hereditary lipoprotein lipase deficiency (hereditarylipoprotein LIPASE DEFICIENCY, LPLD), spinal muscular atrophy (spinal muscular atrophy, SMA); there are various genetic diseases such as Du's muscular dystrophy (Duchenne muscular dystrophy, DMD) and the like. The limited packaging capacity of adeno-associated viral vectors (less than 4.7 kb) is one of the main reasons limiting their clinical use. Dual vector AAV is one of the approaches to address the limited packaging capacity of adeno-associated viral vectors, with the most popular studies of protein-level splicing reactions employing intein trans-splicing. The application of the intein trans-splicing mainly refers to that an independent expression frame is formed by an N-terminal protein sequence of one protein and an N-terminal sequence of the intein, and another independent expression frame is formed by a C-terminal protein sequence of the protein and a C-terminal of the intein, and when the two are expressed in cells at the same time, a shearing and splicing reaction can be carried out through the specific combination of the protein to form a mature protein. The method has high splicing reaction efficiency and can also maintain the activity of the protein, so the method is widely applied. At present, intein Npu DnaE(Nostoc Punctiforme DnaE)(Zettler J,Vivien Schütz,MootzH D.The naturally split Npu DnaE intein exhibits an extraordinarily high rate in theprotein trans-splicing reaction[J].Febs Letters,2009,583(5):909-914.DOI:10.1016/j.febslet.2009.02.003.),Cfa(Engineered-consensus)(Stevens,Adam J,Brown,et al.Design of a Split Intein with Exceptional ProteinSplicing Activity[J].2023.10.9),Gp41-1(Cyanophage-like)(Bareket D,Nir L,Stoddard B L,et al.Fractured genes:a novel genomic arrangement involving newsplit inteins and a new homing endonuclease family[J].Nucleic Acids Research,2009,37(8):2560.) is widely applied to double-vector AAV design construction. However, the dual vector AAV only solves the expression problem of genes smaller than 9kb, and for genes larger than 9kb, it remains a challenge for clinical trials.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide an intein combined carrier and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect, the present invention provides an intein combination vector comprising a first expression vector, a second expression vector, a third expression vector;

The first expression vector comprises a promoter sequence, a target polypeptide N-terminal sequence and a first intein N-terminal sequence;

the second expression vector comprises a promoter sequence, a first intein C-terminal sequence, a target polypeptide intermediate sequence and a second intein N-terminal sequence;

The third expression vector comprises a promoter sequence, a second intein C-terminal sequence and a target polypeptide C-terminal sequence.

Wherein the first intein and the second intein are two inteins that do not undergo a protein trans-cleavage reaction with each other. The insertion position of the target polypeptide can be regulated and combined according to the target polypeptide sequence.

The intein combination of the present invention, as shown in FIG. 1, is designed to not only take into account the differences in splicing efficiency and efficiency of cleavage due to the insertion position of the intein, but also avoid erroneous splicing due to cross-reaction between the inteins. The three expression vectors of the intein combined vector are simultaneously introduced into cells or organisms, and independent three peptide segments can be spliced into a complete target polypeptide sequence through the trans-shearing splicing effect of the intein. The intein combined vector provides a solution for the expression of long polypeptides of vectors limited by loading capacity, such as adeno-associated virus vectors, and has high splicing efficiency of carrier polypeptide shearing, and no cross reaction exists between inteins, so that the incorrect splicing between target polypeptide fragments is avoided, and the problem of delivering genes larger than 9kb can be solved. Has important significance in gene therapy, polypeptide production and other aspects.

As a preferred embodiment of the intein combination carrier of the present invention, the first intein or the second intein is any one of Npu DnaE (Nostoc Punctiforme DnaE), cfa (Engineered-consensus), gp41-1 (Cyanophage-like).

Preferably, the intein combination carrier of the present invention will NpuDnaE be used in combination with GP41 intein or Cfa is used in combination with GP41 intein. In the practical application process, a plurality of insertion sites can be selected, the shearing and splicing reaction of the target protein can be completed correctly and efficiently in the combined process, and meanwhile, no cross reaction occurs between different inteins, thereby providing an application example and a method design for expressing genes with more than 9kb by using adeno-associated virus vectors.

As a further preferred embodiment of the intein combination carrier according to the present invention, the first intein is Npu DnaE and the second intein is Gp41-1.

As a further preferred embodiment of the intein combination carrier according to the present invention, the first intein is Cfa and the second intein is Gp41-1.

As a preferred embodiment of the intein combination vector of the present invention, the expression vector is any one of an adeno-associated viral vector, a lentiviral vector, and an mRNA delivery vector.

In a second aspect, the invention provides a virus comprising the intein combination vector.

In a third aspect, the present invention provides a host bacterium comprising the virus.

In a fourth aspect, the invention provides a cell comprising the virus.

In a fifth aspect, the present invention uses the intein combination vector, the virus, the host bacterium, the cell in gene therapy or protein production.

Compared with the prior art, the invention has the beneficial effects that:

The intein combined vector can select more insertion sites in the practical application process, has high shearing efficiency, does not have cross mismatch reaction between the inteins, ensures the correct splicing of target polypeptides, and has important significance in the aspects of expression and production of long polypeptides, gene therapy and the like.

Drawings

FIG. 1 is a schematic diagram of an intein-combination vector.

FIG. 2 is a 48-hour fluorescence plot of monogranular virus transfected cells;

In FIG. 2 ,A：EGFP(1-70aa)+NpuDnaE-N;B：NpuDnaE-C+EGFP(71-239aa);C:NpuDnaE-C+EGFP(71-147aa)+GP41-N;D：GP41-C+EGFP(148-239aa);E：EGFP(1-147aa)+GP41-N;F：EGFP(1-147aa)+NpuDnaE-N;G：EGFP(1-70aa)+GP41-N.

FIG. 3 is a C-terminal splice validation of the N-terminal and NpuDnaE-terminal inteins validating GP41 inteins;

In FIG. 3 ,A+B：EGFP(1-70aa)+NpuDnaE-N、NpuDnaE-C+EGFP(71-239aa);G+B：EGFP(1-70aa)+GP41-N、NpuDnaE-C+EGFP(71-239aa).

FIG. 4 is a graph verifying the N-terminal cleavage splice of NpuDnaE intein with the C-terminal cleavage splice of GP41 intein;

In FIG. 4 ,E+D：EGFP(1-147aa)+GP41-N、GP41-C+EGFP(148-239aa);F+D：EGFP(1-147aa)+NpuDnaE-N、GP41-C+EGFP(148-239aa).

FIG. 5 is a diagram showing the fluorescence of NpuDnaE cells in combination with GP 41;

In fig. 5, a+c+d: EGFP (1-70 aa) + NpuDnaE-N, npuDnaE-C+ EGFP (71-147 aa) +GP41-N, GP41-C+ EGFP (148-239 aa).

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.

The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.

The present invention solves the problem of delivery of genes greater than 9kb by intein combination, i.e., the design of three vector AAV use, as shown in FIG. 1. The intein combination not only gives consideration to the shearing and splicing efficiency difference and efficiency caused by the intein insertion position, but also avoids the error splicing caused by cross reaction between the inteins. The intein combination of the present invention is used NpuDnaE in combination with GP41 intein, or Cfa (Cfa intein is optimized from NpuDnaE and has the same properties as NpuDnaE intein) is used in combination with GP41 intein, and any other changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention should be equivalent substitution.

The intein combination amino acid sequences referred to in the examples are shown in Table 1:

The intein amino acid sequence of Table 1

Note that: the underlined part is the intein N-terminal sequence and the bolded part is the intein C-terminal sequence

That is, the N-terminal sequence of NpuDnaE, CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN,, is denoted NpuDnaE-N, SEQ ID NO:1, a step of;

NpuDnaE the C-terminal sequence is IKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN, denoted NpuDnaE-C, SEQ ID NO:2;

the N-terminal sequence of GP41 is CLDLKTQVQTPQGMKEISNIQVGDLVLSNTGYNEVLNVFPKSKKKSYKITLEDGKEIICSEEHLFPTQTGEMNISGGLKEGMCLYVKE, which is marked as GP41-N, SEQ ID NO:3, a step of;

The C-terminal sequence of GP41 is MMLKKILKIEELDERELIDIEVSGNHLFYANDILTHN, denoted GP41-C, SEQ ID NO:4, a step of;

Cfa has the N-terminal sequence CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP,, which is marked as Cfa-N, SEQ ID NO:5, a step of;

Cfa has the C-terminal sequence VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN, designated Cfa-C, SEQ ID NO:6.

Example 1: plasmid construction

The target plasmid is constructed by using the polypeptide as EGFR, npuDnaE and GP41 intein as an example to design a vector and using an equal-molecular tool such as seamless cloning. The following plasmids were synthesized:

TABLE 2 plasmid

The primer was biosynthesized from Jin Weizhi. See in particular table 3.

TABLE 3 primer sequences

(1) Plasmid EGFP (1-70 aa) + NpuDnaE-N was constructed as numbered 1.

1) The EA021 plasmid (pAAV-EGFP) (the EA021 plasmid comprises 5'ITR and 3' ITR sequences at two ends, and a GOI part is formed by connecting CAG Promoter, EGFP, WPRE and SV40 pA in series) is used as a template, the plasmid contains expression elements such as CAG+EGFP, N1-F1/N1-R1 are used as primers, and an EGFP (1-70 aa) fragment is amplified and named as fragment 1.

The PCR reaction system is shown in Table 4:

TABLE 4 reaction system

Composition of the components	Volume of
		Primers N1-F1	1μL
Primers N1-R1	1μL
		Template EA021	50ng
2*PrimeSTAR buffer	25μL
		Deionized water	Supplement to 50. Mu.L

The PCR amplification system is shown in Table 5:

TABLE 5 amplification System

2) The PCR product was subjected to gel recovery to obtain fragment 1. Meanwhile, a plasmid NpuDnaE (purchased from Guangzhou Pi Biotechnology Co., ltd.) containing NpuDnaE intein and N1-F2/N1-R2 as primers are used as templates to amplify NpuDnaE N terminal sequence, and the product is named fragment 2. Plasmid EA021 was cut with BamH I and EcoR I endonucleases and the 4027bp fragment was recovered, the product designated fragment 3.

3) And (3) performing seamless cloning by using Exnase Multis ligase, connecting the vector fragment with a PCR product, and reacting at 37 ℃ for 30 min.

The connection system is shown in Table 6:

Table 6 connection system

4) E.coli DH5 alpha is transfected, the plasmid is coated on an ampicillin plate, the bacteria are picked and detected every other day, positive clones are sent to Guangzhou gold only intelligent company for sequencing, and the plasmid with correct sequencing result is named EGFP (1-70 aa) + NpuDnaE-N.

(2) The same procedure constructs plasmid NpuDnaE-C+ EGFP (71-239 aa) numbered 2.

The NpuDnaE C terminal sequence was amplified using plasmid NpuDnaE as template and N2-F1/N2-R1 as primer, and recovered by gel, and the resulting PCR product was designated as fragment 4. The EGFP (71-239 aa) coding sequence was amplified using the EA021 plasmid as template and N2-F2/N2-R2 as primer, and the resulting PCR product was designated fragment 5 by gel recovery. Fragment 4, fragment 5, and vector recovery fragment 3 were mixed and subjected to seamless cloning, and the properly sequenced plasmid was designated NpuDnaE-C+ EGFP (71-239 aa)

(3) The same procedure constructs plasmid NpuDnaE-C+ EGFP (71-147 aa) +GP41-N, numbered 3.

The NpuDnaE-C+ EGFP (71-147 aa) sequence was amplified using the plasmid numbered 2 as template and N3-F1/N3-R1 as primer, and the product gel was recovered and designated as fragment 6. The N3-F2/N3-R2 was used as a primer, the GP41 plasmid (purchased from Guangzhou Pi Biotechnology Co., ltd.) containing the GP41 intein, the GP41-N terminal sequence was amplified, and the product gel was recovered and designated as fragment 7. Fragment 6, fragment 7, vector recovery fragment 3 were mixed and subjected to seamless cloning, and the properly sequenced plasmid was designated NpuDnaE-C+ EGFP (71-147 aa) +GP41-N.

(4) The same procedure constructs plasmid GP41-C+ EGFP (148-239 aa) numbered 4.

The GP41 plasmid is used as a template, N4-F1/N4-R1 is used as a primer, the GP41-C end sequence is amplified, and the product is named as a fragment 8. Using EA021 as template, primer N4-F2/N4-R2, EGFP (148-239 aa) sequence was amplified and the gel recovery product was designated fragment 9. Fragment 6, fragment 7, vector recovery fragment 3 were mixed for seamless cloning and the properly sequenced plasmid was designated GP41-C+ EGFP (148-239 aa).

(5) The same procedure constructs plasmid EGFP (1-147 aa) +GP41-N, numbered 5.

EGFP (1-147 aa) sequence was amplified using EA021 as template, primer N5-F1/N5-R1, and the gel recovery product was designated fragment 10. The GP41 plasmid is used as a template, the primer N5-F1/N5-R1 is used for amplifying the GP41-N sequence, and the gel recovery product is named as fragment 11. Fragment 10, fragment 11, vector recovery fragment 3 were mixed and cloned seamlessly and the properly sequenced plasmid was designated EGFP (1-147 aa) +GP41-N.

(6) The same procedure constructs plasmid EGFP (1-147 aa) + NpuDnaE-N, numbered 6.

EGFP (1-147 aa) sequence was amplified using EA021 as template, primer N6-F1/N6-R1, and the gel recovery product was designated fragment 12. Using plasmid NpuDnaE as a template, primer N6-F2/N6-R2, npuDnaE-N sequence was amplified and the gel recovery product was designated as fragment 13. Fragment 12, fragment 13, vector recovery fragment 3 were pooled, seamlessly cloned, and the properly sequenced plasmid was designated EGFP (1-147 aa) + NpuDnaE-N.

(7) The same procedure constructs plasmid EGFP (1-70 aa) +GP41-N, numbered 7.

The EGFP (1-70 aa) sequence was amplified using EA021 as template, primer N7-F1/N7-R1, and the gel recovery product designated fragment 14. The plasmid GP41 is used as a template, the primer N7-F2/N7-R2 is used for amplifying the GP41-N sequence, and the gel recovery product is named as fragment 15. Fragment 14, fragment 15, vector recovery fragment 3 were mixed and cloned seamlessly and the properly sequenced plasmid was designated EGFP (1-70 aa) +GP41-N.

Example 2: preparation of recombinant adeno-associated virus

In order to better verify NpuDnaE intein and GP41 intein combinations and whether there are N-terminal and C-terminal mismatches in different inteins, 7 plasmids designed in example 1 were individually subjected to adeno-associated virus packaging, 293T cells were infected by different adeno-associated virus combinations, and whether EGFP was expressed smoothly was observed to determine the cleavage splice of the inteins in the cells.

The recombinant adeno-associated virus package is specifically:

Cells were seeded at a density of 5E+5cell/ml in 15cm dishes and incubated overnight for 16-18 hours. 15 μg pHelper, 10 μg pRep2CapDJ, 7 μg plasmid EGFP (1-70 aa) + NpuDnaE-N numbered 1 (or plasmid numbered 2, plasmid numbered 3, plasmid numbered 4, plasmid numbered 5, plasmid numbered 6, plasmid numbered 7) were added per dish. And 10. Mu.g of the transfection reagent polyethylenimine were incubated for transfection, 72 hours after transfection. Cells and supernatant were collected and centrifuged by iodixanol density gradient. Viral titers were determined using SYBRGreenI qPCR and stored in a refrigerator at-80℃prior to use, and the corresponding plasmids were given in Table 7 for virus numbers:

TABLE 7 Virus numbering

Example 3: virus infection assay

Instead of the N-terminus of the same intein, the C-terminus may have the potential to cleave the splice erroneously. To illustrate that the plasmid prepared in example 1 only belongs to the N-terminus of the same intein, the C-terminus can be spliced by cleavage within the cell, whereas the N-terminus and C-terminus of inteins of different origins cannot be spliced by cleavage, the following virus infection assay was performed:

(1) The packaged viruses of example 2 were split at 1E+13GC/ml and individually subjected to cell infection to verify that the designed elemental particle virus itself had fluorescence interference.

The specific experimental steps are as follows: 293T cells with good growth state are subjected to pancreatin digestion, and a proper amount of cell culture medium containing 10% bovine serum is added for neutralization. Cells were collected by centrifugation and were added to 3-5mL of cell culture medium for blow suspension. A small amount of the cell fluid was aspirated for cell counting. 12-well plate cell plating was performed at 2E+5 cells/well. The cells were incubated overnight at 37 ℃. Cells were observed every other day for growth and virus was added to the cells at a titre of moi=1e+6. At 24-48 hours intervals, the fluorescent expression of the cells was observed and recorded by photographing.

As shown in FIG. 1, the cells infected by different simple substance granulocyte adeno-associated viruses for 48 hours have no fluorescence, which indicates that incomplete EGFP proteins (1-70 aa,71-239aa,1-147aa,148-239aa,71-147aa or 148-239 aa) with different lengths expressed by single plasmid viruses can not emit green light under the condition of blue light irradiation.

(2) The N-terminus of GP41 intein and the C-terminus of NpuDnaE intein were verified for the presence of a splice.

The specific experimental steps are as follows: the following groups were first set (see table 8), 2e+5cells/well were plated in 12-well plates and virus was added every other day at a titre of moi=1e+6. The cells were observed for fluorescence for 24-48 hours and photographed for recording.

Table 8 test group

As shown in FIG. 3, the experiment results show that the EGFP (1-70 aa) + NpuDnaE-N, npuDnaE-C+EGFP (71-239 aa) co-infects cells in the group A+B, 48 hours, and obvious fluorescence can be seen, so that the N-terminal and C-terminal sequences of the intein NpuDnaE are placed on different two virus vectors, and the shearing and splicing can be correctly performed in the cells. At 48 hours, the G+B group, EGFP (1-70 aa) +GP41-N, npuDnaE-C+EGFP (71-239 aa), co-infects cells and fluorescence was not visible. It was revealed that even in the same cell, the GP41-N terminal and NpuDnaE-C were not able to perform the correct cleavage splice.

(3) And verifying whether the N end of NpuDnaE intein and the C end of GP41 intein are subjected to shearing splicing or not.

The specific experimental steps are as follows: the following groups were first set (see table 9), 2e+5cells/well were plated in 12-well plates and virus was added every other day at a titre of moi=1e+6. The cells were observed for fluorescence for 24-48 hours and photographed for recording.

Table 9 test group

As shown in FIG. 4, the E+D group, EGFP (1-147 aa) +GP41-N, GP41-C+EGFP (148-239 aa), co-infects cells, and it is evident that fluorescence was observed. Indicating that GP41 intein can be correctly spliced in cell by shearing. The F+D group, EGFP (1-147 aa) + NpuDnaE-N, GP41-C+EGFP (148-239 aa), co-infects cells and fluorescence is not visible. It is stated that NpuDnaE-N and GP41-C cannot be spliced by shearing.

As can be seen from the above experiments, single virus infects cells or co-infects cells at the N-terminus and C-terminus of different inteins, and the splicing and splicing of protein peptide fragments cannot be performed correctly in the cells, and only co-infects cells at the N-terminus and C-terminus of the same intein, such as NpuDnaE-N and NpuDnaE-C or GP41-N and GP41-C, can perform correct splicing and splicing.

(4) EGFP was divided into three peptide fragments, EGFP (1-70 aa), EGFP (71-147 aa) and EGFP (148-239 aa), which were linked correctly in cells by NpuDnaE to the GP41 intein.

The specific experimental steps are as follows: the cells were co-infected with the A+C+D virus, EGFP (1-70 aa) + NpuDnaE-N, npuDnaE-C+EGFP (71-147 aa) +GP41-N, GP41-C+EGFP (148-239 aa), fluorescence was observed for 24-48 hours, and photographed for recording.

As shown in FIG. 5, it is clear from the graph that the co-infected cells of the A+C+D groups have obvious fluorescence expression, and the experiments of the combination (1), (2) and (3) show that NpuDnaE can be combined with GP41, and NpuDnaE and GP41 intein co-infect cells, and that the shearing splice between peptide fragments does not have mismatch.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. An intein combination vector, characterized in that the intein combination vector comprises a first expression vector, a second expression vector and a third expression vector;

The first expression vector comprises a promoter sequence, a target polypeptide N-terminal sequence and a first intein N-terminal sequence;

the second expression vector comprises a promoter sequence, a first intein C-terminal sequence, a target polypeptide intermediate sequence and a second intein N-terminal sequence;

The third expression vector comprises a promoter sequence, a second intein C-terminal sequence and a target polypeptide C-terminal sequence.

2. The intein combination vector of claim 1, wherein the first intein and the second intein are inteins that do not undergo a protein trans-cleavage reaction with each other.

3. The intein combination vector of claim 1, wherein the first intein or the second intein is any one of Npu DnaE, cfa, gp 41-1.

4. The intein combination vector of claim 3, wherein the first intein is Npu DnaE and the second intein is Gp41-1.

5. The intein combination carrier of claim 3, wherein the first intein is Cfa and the second intein is Gp41-1.

6. The intein combination vector of claim 1, wherein the expression vector is any one of an adeno-associated viral vector, a lentiviral vector, an mRNA delivery vector.

7. A virus comprising the intein combination vector of any one of claims 1-6.

8. A host bacterium comprising the virus according to claim 7.

9. A cell comprising the virus of claim 7.

10. Use of the intein combination vector of any one of claims 1-6, the virus of claim 7, the host bacterium of claim 8, the cell of claim 9 in gene therapy or protein production.