Chimeric vector and preparation method and use thereof

Abstract

A chimeric vector is provided in the present invention, which is formed by ligating a Vif protein and a functional protein, the functional protein being a Raf protein or a Rev protein. By designing and constructing a Rev-Vif-C vector and then demonstrating that the Rev-Vif-C vector has a good anti-virus effect by a variety of experiments, the present invention proposes a novel anti-virus technology against the Rev protein of HIV-1. Moreover, by designing and constructing a RBD-Vif-C vector and then demonstrating that the RBD-Vif-C vector has a good tumor cell killing effect by cell-level experiments in vitro and experiments in vivo with nude mouse tumor models, the present invention proposes a novel anti-tumor technology specifically against mutant KRAS.

Claims

1. A fusion protein comprising a Rev or functional fragment thereof directly fused to a Vif protein lacking its N-terminal amino acids at positions 1-79 of SEQ ID NO:5.

2. The fusion protein of claim 1, wherein the N-terminal amino acids of Vif at positions 1-79 of SEQ ID NO: 5 are replaced with at least one oligomerization domain of the Rev protein.

3. The fusion protein of claim 2, wherein the at least one oligomerization domain of the Rev protein is the N-terminal oligomerization domain of the Rev protein.

4. The fusion protein of claim 3, wherein the N-terminal oligomerization domain of the Rev protein comprises amino acids 9-26 of SEQ ID NO: 6.

5. The fusion protein of claim 2, wherein the at least one oligomerization domain of the Rev protein is the C-terminal oligomerization domain of the Rev protein.

6. The fusion protein of claim 5, wherein the C-terminal oligomerization domain of the Rev protein comprises amino acids 51-65 of SEQ ID NO: 7.

7. The fusion protein of claim 2, wherein the at least one oligomerization domain of the Rev protein is both the N-terminal and the C-terminal oligomerization domains of the Rev protein.

8. The fusion protein of claim 7, wherein the N-terminal and C-terminal oligomerization domains of the Rev protein comprise amino acids 9-65 of SEQ ID NO: 8.

9. A composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier.

10. A nucleic acid molecule encoding the fusion protein of claim 1.

11. An expression vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the expression vector of claim 11.

13. A method for making a fusion protein comprising a Rev or functional fragment thereof directly fused to a Vif protein lacking its N-terminal amino acids at positions 1-79 of SEQ ID NO:5, said method comprising: culturing the host cell of claim 12 under conditions that result in the production of a fusion protein comprising a Rev or functional fragment thereof directly fused to a Vif protein or a functional fragment thereof; and isolating the fusion protein from the host cell culture.

14. A fusion protein comprising a Raf or functional fragment thereof directly fused to a Vif protein lacking its N-terminal amino acids at positions 1-79 of SEQ ID NO:5.

15. The fusion protein of claim 14, wherein the N-terminal amino acids of Vif at positions 1-79 of SEQ ID NO: 5 are replaced with a binding domain of an N-terminus of the Raf protein which can specifically bind to GTP-Kras.

16. A composition comprising the fusion protein of claim 14 and a pharmaceutically acceptable carrier.

17. A nucleic acid molecule encoding the fusion protein of claim 14.

18. An expression vector comprising the nucleic acid molecule of claim 17.

19. A host cell comprising the expression vector of claim 18.

20. A method for making a fusion protein comprising a Raf or functional fragment thereof directly fused to a Vif protein lacking its N-terminal amino acids at positions 1-79 of SEQ ID NO:5, said method comprising: culturing the host cell of claim 19 under conditions that result in the production of a fusion protein comprising a Raf or functional fragment thereof directly fused to a Vif protein or a functional fragment thereof; and isolating the fusion protein from the host cell culture.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the construction model of the chimeric vector RBD-Vif-C.

(2) FIG. 2 illustrates that the protein encoded by the chimeric vector RBD-Vif-C can degrade the KRAS protein and inhibit its signaling pathway of phosphorylation in the downstream.

(3) FIG. 3 illustrates that the protein PTD-RBD-Vif-C has a good killing effect on the tumor cells in vitro.

(4) FIG. 4 illustrates that the protein PTD-RBD-Vif-C has a good anti-tumor effect in mice.

(5) FIG. 5 illustrates that the acute toxicity test of the protein PTD-RBD-Vif-C in mice.

(6) FIG. 6 illustrates the construction model of the chimeric vector Rev-Vif-C.

(7) FIG. 7 illustrates that the protein encoded by the chimeric vector Rev-Vif-C degrades the protein Rev by the ubiquitination pathway.

(8) FIG. 8 illustrates that the protein encoded by the chimeric vector Rev-Vif-C inhibits the replication of multiple viral strains of wild-type HIV-1 by inhibiting the nuclear export function of the Rev-RRE.

DETAILED DESCRIPTION OF THE PREFERED EMBODIMENT

(9) The present invention will be further described in detail in combination with the accompanying drawings and specific embodiments below. Unless otherwise specified, reagents, equipment and methods used in the present invention are conventionally commercial reagents, equipment and routinely used methods in the present technical field.

Embodiment 1 A Construction Model of the Chimeric Vector RBD-Vif-C

(10) As is well-known, the occurring of various tumors such as pancreatic cancer, lung cancer and colorectal cancer is greatly related to mutation of the KRAS gene. A single point mutation of KRAS is sufficient to lead to occurrence of a tumor, wherein the mutation of an amino acid at position 12 of the KRAS accounts for 98% of the single point mutation of KRAS. Among the mutation types of the amino acid at position 12, G12V and G12D mutations are the most significant, which accounts for 30% and 51% respectively. Specific degradation of mutant KRAS protein is realized by a degradation mechanism of target protein induced by Vif, and thus a new type of anti-tumor medicine is developed.

(11) In accordance with the literatures, it is known that there is a KRAS binding domain RBD specifically binding to the mutant KRAS on an N-terminus of a RAF-1 protein. Such binding domain can specifically bind to the mutant KRAS protein in vivo and vitro, and its binding affinity for GTP-Kras is 100 times different from that for GDP-Kras. Hence, nucleic acid encoding the N-terminus of the Vif protein is replaced with nucleic acid encoding the RBD, and then it is ligated to the vector of pcDNA3.1 to form a chimeric vector RBD-Vif-C.

(12) Specific steps are provided below:

(13) (1) preparing a chimeric vector, in which nucleic acid encoding an amino acid sequence at positions 1-79 of the N-terminus of the Vif protein is replaced with nucleic acid encoding the RAS binding domain RBD which is provided on a protein structure of Raf and specifically binds to GTP-KRAS, thus a new chimeric vector RBD-Vif-C is constructed;
(2) cloning the chimeric vector into the expression vector pcDNA3.1 and performing a transient expression by transfection;
(3) synthesizing nucleic acid encoding a transmembrane peptide segment PTD and then ligating it to an Escherichia coli expression vector of pet-32a;
(4) performing a PCR amplification using the RBD-Vif-C as a template, ligating the obtained PCR product RBD-Vif-C to the expression vector pet-32a, and nucleic acid encoding the PTD being located on the N-terminus of the RBD-Vif-C to form a fusion expression vector PTD-RBD-Vif-C;
(5) transforming plasmids of the PTD-RBD-Vif-C into the Escherichia coli BL21(DE3);
(6) culturing the transformed Escherichia coli;
(7) a single PTD-RBD-Vif-C protein is obtained after a purification using a nickel column.

(14) The construction of the vector mentioned in step (1) comprises steps as follows: synthesizing two pairs of primers according to nucleic acid encoding Vif protein of HIV-1 and KRAS protein respectively, then performing the PCR amplification with the aforementioned primers using a sequence of PNL4-3 plasmids of HIV-1 virus as the template; then cloning the PCR amplification product into the pcDNA3.1 by different enzyme cutting sites, wherein nucleic acid encoding segment of RBD is inserted into nucleic acid encoding the N-terminus of the Vif.

(15) Culturing mentioned in step (6) is to inoculate a single colony of the transformed Escherichia coli to a LB liquid medium containing ampicillin for cultuting overnight; and then the colony is inoculated to the 37 C. preheated LB liquid medium containing ampicillin with a volume ratio of 1:50 for culturing until OD600 reaches 0.6; IPTG is added until having a final concentration of 0.4 mmol/L, the PTD-RBD-Vif-C protein expresses, and the bacteria is collected after induction.

(16) A method of purification mentioned in step (7) is to wash total bacteria with PBS Buffer. After being resuspended and ultrasonic treated, a lysate supernatant is obtained by a high speed centrifugation, and an eluted protein is collected after an affinity chromatography purification using the nickel column.

Embodiment 2 the Protein Encoded by the Chimeric Vector RBD-Vif-C can Degrade the KRAS Protein and Inhibit its Signaling Pathway of Phosphorylation in the Downstream

(17) The mutant KRAS genes, KRAS-G12D and KRAS-G12V, are fusion expressed with the RFP gene respectively and are cloned into the vector of pcDNA3.1. The expression of the KRAS can be reflected by observing the expression of the RFP. Meanwhile, the expression of the KRAS is further verified by Western Blot.

(18) (1) Co-transfecting the vector including nucleic acid encoding the KRAS-G12D-RFP with the vector RBD-Vif-C or the KRAS-G12V-RFP with the vector RBD-Vif-C in 293t cells in six-well plates, with a weight of plasmids of 1 g; 48 hours after co-transfection, observing the expression of the RFP; meanwhile collecting a cell lysate for detecting the expression of the KRAS by the Western Blot.

(19) The experiment shows that the RBD-Vif-C can degrade the mutant KRAS protein.

(20) (2) Co-transfecting the vector including nucleic acid encoding the KRAS-G12D and the plasmid of the RBD-Vif-C in 293t cells in six-well plates; 48 hours later, collecting the cell lysate for detecting the expressions of ERK and phosphorylated ERK by the Western Blot.

(21) The experiment shows that the RBD-Vif-C can inhibit its signaling pathway of phosphorylation in the downstream of the KRAS protein by degrading it.

Embodiment 3 Protein PTD-RBD-Vif-C has a Good Killing Effect on the Tumor Cells In Vitro

(22) In consideration of factors such as low efficiency of the transfection in cell plasmid system of pancreatic cancer, lung cancer and colorectal cancer, and subsequent druggability factor of protein, hence, Escherichia coli is chosen to express the corresponding RBD-Vif-C protein while GFP-Vif-C is chosen as a negative control protein, in order to attempt to verify the inhibition of the expression of KRAS gene by the RBD-Vif-C by means of the action mode of protein directly.

(23) Firstly with reference to the methods from the related literatures, an effective transmembrane oligopeptide PTD is synthesized and the sequence thereof is shown in the drawings. Then nucleic acid encoding the transmembrane peptide and the vector RBD-Vif-C are cloned into a prokaryotic expression vector of pET-32a together, expressing the RBD-Vif-C protein by a prokaryotic system. The PTD-RBD-Vif-C protein with His-tag is purified by a nickel column and the protein with relatively high purity can be obtained after several optimizations, as shown in FIGS. 3-13. Further, endotoxin in the protein is removed by the method of Triton X-114. Since the protein itself has the transmembrane peptide, the purified protein can be directly added into the cell culture supernatant, which provides the subsequent experiments with greater convenience.

(24) (1) Planking multiple cells into 24-well plates, after the cells being adhered; adding 4 g/well of the PTD-RBD-Vif-C protein or the PTD-GFP-Vif-C protein respectively; after treating the cells for 48 hours, observing and recording growth states of the various cells with a microscope.

(25) The experiment shows that PTD-RBD-Vif-C protein can inhibit the proliferation of various tumor cells related to the KRAS.

(26) (2) Adding 0 g/well, 2 g/well, 4 g/well and 8 g/well of the PTD-RBD-Vif-C protein to the Panc-1 cells and the A549 cells in the 24-well plates respectively; collecting the cells after treating them for 48 hours; labeling flow antibody of Annexin-V FITC and detecting the apoptosis of these two kinds of cells.

(27) The experiment shows the PTD-RBD-Vif-C protein can induce the apoptosis of tumor cells related to the KRAS and has a certain concentration gradient dependency.

(28) (3) Adding 2 g/well of the PTD-GFP-Vif-C protein and 2 g/well of the PTD-RBD-Vif-C protein to the Panc-1 cells and the A549 cells in six-well plates respectively; after treating them for 12 hours, collecting cell lysate for detecting the expression of endogenous KRAS of these two kinds of cells by Western Blot.

(29) The experiment shows that the PTD-RBD-Vif-C protein can inhibit the expression of the endogenous KRAS gene.

Embodiment 4 PTD-RBD-Vif-C Protein has a Good Anti-Tumor Effect in Mice

(30) Six male Balb/c nude mice of 4-6 months old were ordered from the Animal Experimental Center of Sun Yat-sen University. They were divided into two groups randomly and each group contained three mice. The mice were subcutaneously inoculated with 110.sup.6 of Panc-1 cells and A549 cells respectively to generate tumors. Two weeks later, subcutaneous generation of tumor in mice was observed, and the PTD-RBD-Vif-C protein and the PTD-GFP-Vif-C protein were started to be injected into the mice 2-3 times a week. Four weeks later, the mice were executed and tumor tissues were taken for observation and were recorded the weight.

(31) The experiment shows that the PTD-RBD-Vif-C protein has a good anti-tumor effect in mice, especially in the tumor cells of pancreatic cancer and lung cancer.

Embodiment 5 the Acute Toxicity Test of the PTD-RBD-Vif-C Protein in Mice

(32) (1) 18 male Balb/c mice of 4-6 months old were ordered from the Animal Experimental Center of Sun Yat-sen University. They were divided into 3 groups randomly and each group contained 6 mice.

(33) (2) The PTD-RBD-Vif-C protein was injected into the mice by means of tail vein injection with an injection dose of 0 mg/kg (PBS), 10 mg/kg and 20 mg/kg respectively, each group contained 6 mice.

(34) (3) Two weeks later, blood samples of the mice were taken for detection of liver function and renal function, and the experiment result is noimal.

(35) (4) After execution, each tissue samples of heart, liver, spleen, lung and kidney were taken from the mice to perform HE staining, in order to observe whether there was a lesion in the organ or not.

(36) The experiment shows that 20 mg/kg dose of protein is non-toxic and safe for the mouse, and such protein has a better druggability.

Embodiment 6 the Construction Model of the Chimeric Vector Rev-Vif-C

(37) Nucleic acid encoding two oligomerization binding domains (amino acids at positions 9-26 and amino acids at positions 51-65) on the HIV-1 Rev gene were respectively cloned to the N-terminus of the Vif gene, replaced nucleic acid encoding the binding sites (amino acids at positions 1-79) of APOBEC3G, and then were ligated to the vector of pcDNA3.1 to form three different chimeric vectors, which are named as ROL1-Vif-C, ROL2-Vif-C and ROL12-Vif-C respectively. In particular, ROL1 represents nucleic acid encoding the oligomerization binding domain of the N-terminus of the Rev, i.e. the amino acids at positions 9-26; ROL2 represents nucleic acid encoding the oligomerization binding domain of the C-terminus of the Rev, i.e. the amino acids at positions 51-65; and ROL12 represents nucleic acid encoding the two oligomerization binding domains tandem the N-terminus and C-terminus of the Rev, i.e. the amino acids at positions 9-26 and amino acids at positions 51-65.

(38) Specific steps are as follows:

(39) (1) Respectively replacing nucleic acid encoding the amino acid sequence at positions 1-79 of the N-terminus of the Vif protein with nucleic acid encoding two oligomerization domains provided on the protein structure of the Rev, thus three new chimeric vectors ROL1-Vif-C (containing nucleic acid encoding the oligomerization domain of the N-terminus of the Rev, i.e. the amino acids at positions 9-26), ROL2-Vif-C (containing nucleic acid encoding the oligomerization domain of the C-terminus of the Rev, i.e. the amino acids at positions 51-65) and ROL12-Vif-C (containing nucleic acid encoding two oligomerization domains of the N-terminus and C-terminus of the Rev, i.e. the amino acids at positions 9-65) were constructed.

(40) (2) Cloning these three chimeric vectors into the expression vector pcDNA3.1 and performing a transient expression by transfection.

(41) In particular, the construction of the vectors mentioned in step (1) comprises steps as follows: synthesizing 4 pairs of primers according to nucleic acid encoding the Vif protein and the Rev protein of HIV-1 respectively, then performing PCR amplification with the aforementioned primers using the sequence of the PNL4-3 plasmids of HIV-1 virus as template; then cloning the PCR amplification product into the pcDNA3.1 by different enzyme cutting sites, wherein nucleic acid encoding segment of the Rev is inserted into nucleic acid encoding the N-terminus of the Vif segment.

Embodiment 7 the Protein Encoded by the Chimeric Vector Rev-Vif-C Degrades Rev Protein by the Ubiquitination Pathway

(42) HIV-1 Rev gene was performed a fusion expression with the RFP gene and they were cloned into the vector of pcDNA3.1, thus the expression of the Rev can be reflected by observing the expression of the RFP; meanwhile the Rev gene was performed the fusion expression with a HA label in order to further verify the expression of Rev by the Western Blot.

(43) (1) Co-transfecting the vector including nucleic acid encoding the Rev-RFP and the vector ROL1-Vif-C (or the ROL2-Vif-C or the ROL12-Vif-C) in the 293t cells in the 24-well plates with an amount of plasmids of 1:0, 1:1, 1:2 and 1:3 respectively; 48 hours after transfection, observing the expression of the RFP; meanwhile, co-transfecting the vector including nucleic acid encoding the Rev-HA and the vector ROL1-Vif-C in the 293t cells on the six-well plates with the amount of plasmids of 1:0, 1:1, 1:2 and 1:3 respectively; 48 hours after transfection, collecting the cell lysate for detecting the expression of Rev by the Western Blot.

(44) The experiment shows that the proteins encoded by the three chimeric vectors can inhibit the expression of Rev protein and have a certain concentration gradient dependency.

(45) (2) Co-transfecting 200 ng of the vector including nucleic acid encoding Rev-RFP and 200 ng of ROL12-Vif-C vector in the 293t cells in the 24-well plates; 24 hours later, adding 10 M of MG132 to treat the cells; after treating them for 12 hours, observing the expression of the RFP.

(46) (3) Co-transfecting 200 ng of the vector including nucleic acid encoding Rev-RFP and 200 ng of ROL12-Vif-C vector in the 293t cells in the 24-well plates; 6 hours later, transfecting 50 nM of si-NC, si-ElonginB, si-ElonginC and si-Culin5; 48 hours after transfection, observing the expression of the RFP.

(47) (4) Co-transfecting 3 g of Ub-Flag of the plasmid, 3 g of the vector including nucleic acid encoding Rev-HA and 2 g of ROL1-Vif-C, ROL2-Vif-C, ROL12-Vif-C vectors respectively in the 293t cells in the 6 cm plate; 48 hours after transfection, collecting the cell lysate for Co-IP to enrich the Rev-HA protein, and further detecting the ubiquitination of the Rev protein.

(48) The above three experiments show that the protein encoded by the chimeric vectors degrade the Rev protein by the ubiquitination pathway mediated by the Vif.

Embodiment 8 the Protein Encoded by the Chimeric Vector Rev-Vif-C Inhibits the Replication of Various Viral Strains of Wild-Type HIV-1 by Inhibiting the Nuclear Export Function of the Rev-RRE

(49) There is a splicing donor SD and a splicing acceptor SA on PDM628. When the Rev protein is not present, the luciferase gene carried by the PDM628 is cleaved, causing a trace expression of luciferase. When two kinds of plasmids co-transfect, the Rev protein is combined with the RRE element and the luciferase gene segment is brought out of the cell nucleus in order to avoid being cleaved by SD and SA, and thus the luciferase expresses massively. Therefore, when the expression of Rev protein is inhibited, the system of nuclear export related to the Rev-RRE will be inhibited and the amount of expression of luciferase will decrease by this time. Based on such principle, it can be judged whether the function of the Rev protein is affected or not.

(50) (1) Co-transfecting 10 ng of.sub.PDM628 plasmids and 10 ng of Rev plasmids respectively in the 293t cells on 96-well plates, then co-transfecting different concentrations of ROL1-Vif-C, ROL2-Vif-C, ROL12-Vif-C and M10 plasmids; 48 hours after transfection, collecting the cell lysate to detect the expression of luciferase.

(51) The experiment shows that proteins encoded by three chimeric vectors each can inhibit the system of nuclear export related to the Rev-RRE, and have a certain concentration gradient dependency. Meanwhile, the effect of the proteins encoded by the provided chimeric vectors is apparently better than the existing RevM10 mutant.

(52) (2) Co-transfecting 50 ng of PNL4-3 plasmids and different concentrations of ROL1-Vif-C, ROL2-Vif-C, ROL12-Vif-C plasmids (50 ng, 100 ng, 150 ng) respectively in the 293t cells in 24-well plates; 48 hours after transfection, collecting the supernatant to detect the expression of P24 by ELISA. (B), Co-transfecting 50 ng of PYU-2 plasmids and different concentrations of ROL1-Vif-C, ROL2-Vif-C, ROL12-Vif-C plasmids (50 ng, 100 ng, 150 ng) respectively in the 293t cells on the 24-well plate; 48 hours after transfection, collecting the supernatant to detect the expression of P24 by ELISA.

(53) The experiment shows that proteins encoded by three chimeric vectors each can inhibit the replication of various different wild-types HIV-1, and have a certain concentration gradient dependency.