TW201412985A - Protein for regulation of macromolecules into cells and method for regulation of macromolecules into cells - Google Patents

Abstract

The present invention discloses protein for regulation of macromolecules into cells and method for regulation of macromolecules into cells. By IpaB or IpaB803 gene transfection on a cell, IpaB or IpaB803 protein will express in the cell. Followed by co-cultured with a macromolecule, it is found that a macromolecule may enter the cytoplasm or/ and nucleus.

Description

A protein that regulates the entry of macromolecules into cells and a method for regulating the entry of macromolecules into cells

The present invention relates to a method for transporting macromolecules to cells, and more particularly to a method for regulating the entry of macromolecules into cells and the regulation of macromolecules into cells.

According to the eukaryotic cell line, the cell membrane, the nucleus, the intracellular membrane, and the cytosol, etc., wherein the cell membrane is a bilayer phospholipid membrane inside and outside the cell, which is embedded with membrane proteins of various functions and combined with membrane proteins. Sugars form a selective permeable membrane. There are pores on the cell membrane, and the material exchange or information transmission with the surrounding environment, through the pores and the nature of the membrane protein, can regulate the function of the substances entering and leaving the cells inside and outside, in order to maintain the stability within the cells. The nucleus is a closed membranous organelle present in the cell, which contains most of the genetic material in the cell, and the outer part completely covers the nuclear membrane of the nucleus, and separates the intracellular substance from the cytoplasm. The nuclear membrane is also a double-layer phospholipid membrane, which is divided into a core membrane and an outer nuclear membrane, which have nuclear pore complexes (NPCs), a pore size of about 50-100 nm, and a pore diameter of only 9 nm for free diffusion of molecules. It is used to control the entry and exit of substances between the nucleus and the cytoplasm.

Extracellular small molecules can be transported through the pores through the passive transport of diffusion or infiltration, or through the active transport of proteins on the membrane, while macromolecular substances such as bacteria or viruses cannot enter through the cell membrane. In the cell, it is necessary to enter the cell by phagocytosis or pinocytosis of the cell. Small water-soluble molecules or ions in the cytoplasm and proteins less than 40 kDa can enter the nucleus directly through the nuclear membrane by passive transport or diffusion, but larger proteins such as proteins or nucleic acids over 40 kDa need Through the interaction of the nuclear localization signal (NLS) and the input protein α/β (importin α/β) complex, it can enter the nucleus through the nuclear pore.

In detail, the nuclear signal sequence into a plurality of lines of the positively charged basic amino acids lysine (lysine) and arginine (Arginine) composed by (Robbins et al., 1991) , exposed to the surface proteins . Therefore, the substance located in the cytoplasm must carry the nuclear signal itself, and be recognized and interacted by the receptor composed of the input protein α/β on the nuclear membrane, so that the protein with the nucleated cells can be passed through the nuclear pore. Into the nucleus (Lange et al ., 2007). Tobacco Mosaic Virus (TMV) replication enzymes (Figueire et al ., 2002) and shuttle proteins such as HsfA2 protein (Scharf et al., 1998; Heerklotz et al., 2001) have been disclosed in the literature. A sequence with a nuclear signal. Therefore, a macromolecule that does not have a nuclear signal must be assisted by a foreign carrier or external force to enter the cell, and a macromolecule with a nuclear signal can directly enter the cell itself.

At present, in clinical medicine, treatment of diseases such as cancer or hereditary diseases is controlled by drugs or chemotherapy. At present, many studies are devoted to the development of vectors or the use of kits for gene therapy, that is, drugs, Macromolecules such as proteins or deoxyribonucleic acids are directly delivered into the cytoplasm or/and nucleus, so that the substance that is introduced into the nucleus functions in the cell or kills the cell. The vectors currently used can be divided into two types, one is a viral vector, such as a retrovirus, an adenovirus, etc., and the other is a non-viral vector, such as naked DNA, vesicles, etc., but the two Class carriers have their disadvantages as follows:

In the case of viral vectors, the virus can infect cells by itself, which is an efficient base. Due to the transmission system, the pathogenicity or carcinogenicity of the virus itself must be treated before it can be used. Furthermore, the virus system can only be used to carry the target gene, and the size of the target gene is limited, and the use is not only safe. There are some shortcomings, and the scope of application is also limited; in addition, in terms of non-viral carriers, although the safety is better than the viral carrier, the efficiency of delivering the substance is not good, and the practicality and benefit cannot be expected.

In summary, the current carrier system, in addition to the inability to balance safety and practicality, must integrate the substance to be fed into the carrier and then deliver the substance into the target cells. Therefore, the development of a safe and practical transport system that allows macromolecules to enter the cell and play its role is urgently needed by the current biomedical research institute.

The main object of the present invention is to provide a method for introducing macromolecules into cells, comprising the steps of: (A) constructing a recombinant plastid having an exogenous gene, which is used to represent at least one positively charged one. a protein of a basic amino acid sequence, the amino acid sequence encoding KKKL; (B) transducing the recombinant plastid into a eukaryotic cell, and allowing the foreign gene to be expressed in the eukaryotic cell; C) selecting a large molecule, co-cultured with the eukaryotic cell in step B; (D) the macromolecule enters the cell, wherein:

The exogenous gene in step A is the IpaB gene or the IpaB gene derivative product.

The macromolecule selected in step D is obtained as a bacterium, such as Shigella flexneri (S. flexneri ), Escherichia coli, etc., into the cytoplasm and/or nucleus of the eukaryotic cell.

Another object of the present invention is to provide a protein for regulating the entry of macromolecules into cells, wherein the amino acid sequence comprises at least a positively charged basic amino acid sequence. KKKL. Further, the protein that regulates the entry of the macromolecule into the cell is expressed by the IpaB gene or the IpaB gene-derived product.

The invention will be further illustrated by the following examples in conjunction with the drawings.

The abbreviation of the biological material used in the following examples of the present invention is further explained.

The IpaB protein is an abbreviation for Invasion plasmid antigen B, which is secreted by the genus of the genus Fusarium via the third-type secretory system. The IpaB protein is a transposon of the IpaB gene, which is 62.1 kDa (580 amino acids), while the IpaB gene is located on the virulence of the ipa-mxi-spa (invasion plasmid antigen, Membrane expression of Ipa and Surface presentation of In the Ipa Antigens region, the gene length is 1743 bp.

The IpaB protein used in the present invention is represented by the wild type strain SH2308 of Fusarium, wherein the wild type strain SH2308 is a strain which the inventor of the present invention found in the previous study to express the IpaB protein, and The IpaB gene of strain SH2308 was sequenced, and the nucleotide sequence was obtained as SEQ ID NO: 1. . The IpaB gene of strain SH2308 and have been published in the US National Center for Biotechnology Information (National Center for Biotechnology Information; NCBI ) two of Freund's bacillus (S.flexneri) toxicity plasmid (Accession: NC_004851.1; Accession: NC_002698 . 1) IpaB gene alignment in the 1st, found that the position of the 52nd and 507th bases changed from A to T and from T to A, respectively, that is, the difference in the 52nd base position makes the translated amino acid from Su The amino acid (Threonine) becomes alanine, and the difference at the 507th base position does not cause the amino acid to be translated to be different. The above difference does not affect the IpaB protein used in the present invention and the above-mentioned two IpaB proteins, and the 175-178 amino acid sequence of both is KKKL, which is a positively charged basic amino acid sequence.

The IpaB803 protein is obtained by translating the IpaB gene used in the present invention from the ATG start codon to the 803 bp nucleotide fragment, wherein the IpaB803 nucleotide sequence is encoded as SEQ ID NO: 2, which is translated. The IpaB803 protein line also contains a positively charged basic amino acid sequence encoding KKKL.

SHB2308 is a knock-out strain of the IpaB gene of the wild type strain SH2308 of Fusarium.

HCT116 is a human rectal cancer cell line.

pFLAG-CMV2 is a mammalian expression vector containing a CMV2 promoter and a flag sequence (FLAG), wherein the flag sequence binds to the 5' end of the plastid and becomes a It can be expressed in a protein carrier with a N-terminal flag and a histone (C) at the C-terminus.

pFLAG-CMV2-IpaB, which is a recombinant plastid constructed by genetic engineering technology, contains the IpaB gene.

pFLAG-CMV2-IpaB803, which is a recombinant plastid constructed by genetic engineering technology, contains the IpaB803 gene.

Example 1: Construction of recombinant IpaB gene expression plastid

A product containing the IpaB gene fragment was amplified by polymerase chain reaction (PCR), and the restriction product XbaI, SmaI was used to cut the product containing the IpaB gene fragment and the expression vector pFLAG-CMV2, and the adhesion reaction was carried out. (ligasion), a recombinant plastid is obtained, as shown in the first figure.

Example 2: Constructing the expression plastid of recombinant IpaB803 gene

The procedure is the same as in the first example. First, a product containing the IpaB803 gene fragment is amplified by a polymerase chain reaction, and the product containing the IpaB803 gene fragment and the expression vector pFLAG-CMV2 are cut and bound by restriction enzymes XbaI and SmaI. The reaction was carried out to obtain a recombinant plasmid pFLAG-CMV2-IpaB803, as shown in the second figure.

Example 3: Culture HCT116 cell line

The frozen HCT116 cell line was thawed in a 37 ° C water bath, and the cell liquid was aspirated and added to a 10 cm culture plate containing 10 ml of the medium, and cultured in a 37 ° C, 5% carbon dioxide incubator for 24 hours. Remove the 10 cm culture plate, remove the medium, add fresh medium, and return to the incubator to continue the culture until the cells grow to about 8-9 minutes. The medium is removed and 5 ml of the phosphate buffer (Phosphate) is returned. Buffered Saline, PBS) rinse the cells. Remove the phosphate buffer. Add 1 ml of 0.25% trypsin-ethylenetetraminetetraacetic acid (Trypsin-EDTA, SIGMA, Cat. No. T4799), let stand for 1 to 2 minutes at 37 ° C to suspend the cells, and then add 4 ml of medium to stop trypsin - The reaction of ethylenetetraamine tetraacetic acid, and then the cells are broken up to form a cell suspension.

The ratio of the cell suspension to the medium was 1:3 to 1:5, and the total volume was 8 ml. The whole volume was added to a 10 cm culture plate and placed in an incubator for subculture until 8 to 9 minutes. The cells are then subcultured to at least passage 3 in the same manner as described above for use in subsequent examples.

Example 4: Preparation of pFLAG-CMV2 transfected HCT116 cell line

First, a transfer solution was prepared according to the product specification of lipofectamine 2000 (Invitrogen, Cat. No. 11668-027), and was used.

Take a 1.5 ml microcentrifuge tube, add 2 μL of pFLAG-CMV2 plastid DNA at a concentration of 1 μg/μL, and add 48 μL of serum-free cell culture medium (Opti-MEM® I reduced serum medium, Gibco, Cat. No). .31985-070), a DNA solution was obtained.

Another tube of microcentrifuge tube was added, 16 μL of the transfer solution (lipofectamine 2000, Invitrogen) was added, and 34 μL of serum-free cell culture medium (Opti-MEM® I reduced serum medium, Gibco, Cat. No. 31985) was added. -070), a lipofectamine mixture was obtained and allowed to stand at room temperature for 5 minutes.

After the lipofectamine mixture was all added to the DNA solution, it was uniformly mixed and allowed to stand at room temperature for 20 minutes.

The subcultured HCT116 cells in Example 3 were diluted with the medium to a concentration of 1.0 x 10 ⁵ cells/mL. Take a 30 cm culture plate, put two slides, add 1 ml of medium, add 1 ml of cell suspension, put into the incubator, incubate for 48 hours, then add 100 μL of the transfer solution, and culture until the 4th hour. The culture plate was taken out, the supernatant was removed, 2 ml of the culture solution (DMEM) was added, and the culture was continued for a predetermined period of time, and then the pFLAG-CMV2 plasmid was transfected into HCT116 cells for use in the following examples.

Example 5: Preparation of pFLAG-CMV2-IpaB transfected HCT116 cell line

The steps in this example are substantially as described in Example 4, and therefore will not be repeated, except that the DNA solution prepared in this example is the recombinant plasmid DNA of pFLAG-CMV2-IpaB in Example 1. 48 μL of serum-free cell culture medium (Opti-MEM® I reduced serum medium, Gibco, Cat. No. 31985-070) was added to transfect the pFLAG-CMV2-IpaB into the HCT116 cell line.

Example 6: Preparation of pFLAG-CMV2-IpaB803 transfected HCT116 cell line

The steps in this example are substantially as described in Example 4, and therefore will not be described again. However, the DNA solution prepared in this example differs from the recombinant plasmid DNA of pFLAG-CMV2-IpaB803 in Example 2. 48 μL of serum-free cell culture medium (Opti-MEM® I reduced serum medium, Gibco, Cat. No. 31985-070) was added to transfect pFLAG-CMV2-IpaB803 into HCT116 cell line.

Example 7: IpaB gene/IpaB803 gene expressed in HCT116 cells

The transfected HCT116 cells from Example 4 to Example 6 were divided into three groups, wherein the first group was the pFLAG-CMV2 transfected HCT116 cells prepared in Example 4; the second group was the pFLAG prepared in Example 5. - CMV2-IpaB was transfected into HCT116 cells; the third group was transfected with pFLAG-CMV2-IpaB803 prepared in Example VI.

After culturing each group of cells for 24 hours, each group of cells was simultaneously subjected to nuclear fluorescent staining agent (DAPI), anti-FLAG antibody and mitochon fluorescent staining agent (mito-tracker). Fluorescent staining was performed, and cells of each group were observed at different excitation wavelengths by an inverted conjugate focus microscope, and the results are shown in Annexes I to III.

The results of the first to third groups are the results of the first group to the third group. In detail, the A picture of each attachment is the cell type observed under the bright field of view; the B picture of each accessory is the nucleus The result of fluorescent staining of fluorescent staining agent (DAPI), the blue part is the nucleus in each figure; the C picture of each accessory is the result of fluorescent staining of anti-FLAG antibody, green in each figure The site is the detection of IpaB-FLAG fusion protein or IpaB803-FLAG The fusion protein; the D picture of each accessory is the result of fluorescent staining of the mitochon fluorescent dye (mito-tracker), the red part of each figure is a mitochondria; the E picture of each accessory is The results of the overlapping of the fluorescent staining of the B to D of the annex, the blue and green overlapping parts are light blue, indicating that the IpaB-FLAG fusion protein or the IpaB803-FLAG fusion protein enters the nucleus, both red and green. The appearance of the part indicates the expression of the IpaB-FLAG fusion protein or the IpaB803-FLAG fusion protein in the cytoplasm; the F map of each attachment is an enlarged view of the white square in the A of each attachment; the G map of each attachment An enlarged view of the white squares in Figure B of each of the attachments; each of the attachments H is an enlarged view of the white squares in Figure C of each of the attachments; each of the attachments is shown in Figure I. A magnified view of the white box in the figure D; each of the J diagrams of the attachment is an enlarged view of the white box in the E diagram of each attachment; wherein the magnifications of the F to J diagrams of the attachments are respectively 1200 times.

From the results of Annexes I to III, it can be seen that both the IpaB gene and the IpaB803 gene can be transfected into cells and express IpaB protein or IpaB803 protein in the cytoplasm or/and nucleus, respectively.

Example 8: Preparation of SHB2308 bacterial solution

The strain SHB2308 was inoculated on LB medium (LB broth) and cultured overnight at 37 °C. 100 μL of SHB2308 broth was added to a microcentrifuge tube containing 900 μL of M9 solution, serially diluted to 10 ^-7 , and then 100 μL of 10 ^-6 and 10 ^-7 two dilutions of SHB2308 broth was applied to LB medium. The cells were counted, and the remaining SHB2308 broth was stored in a refrigerator at 4 °C. The colony-forming unit (CFU) value was calculated every other day, and the concentration of the original SHB2308 bacteria solution was calculated, and the SHB2308 bacteria solution stored in the refrigerator at 4 °C was taken back to room temperature. Take 1 ml of SHB2308 bacteria solution and centrifuge at 12000 rpm for 5 minutes at room temperature. After removing the supernatant, resuspend the cell pellet in 1 ml of cell culture medium (DMEM) containing no fetal bovine serum. The fetal culture medium was diluted with cell culture medium (DMEM) SHB2308 to a concentration of 1×10 ⁷ CFU/mL for use in the following examples.

Example 9: Preparation of Escherichia coli strain DH5 α bacterial solution

The steps of this example are substantially the same as those described in Example 8, and therefore, no further description is given, except that the present example is obtained by inoculating the Escherichia coli strain DH5α on the medium to obtain an Escherichia coli strain having a concentration of 1×10 ⁷ CFU/mL. The DH5 alpha bacterial solution was used in the following examples, wherein the Escherichia coli strain DH5α was transfected to express enhanced green fluorescent protein (EGFP).

Example 10: Co-cultivating strains SHB2308 and HCT116 cell lines

The cell lines of the transfected plastids prepared in Examples 4 to 6 were respectively divided into three groups, wherein: the first group was pFLAG-CMV2 transfected HCT116 cell line as a control group; the second group was pFLAG- CMV2-IpaB was transfected into HCT116 cell line; the third group was transfected with pFLAG-CMV2-IpaB803 into HCT116 cell line.

The following is the procedure for co-cultivation used in the present example, and the first group is taken as an example for illustration.

First, the cell suspension of the first group was taken, and the cell concentration was diluted to 1.0 x 10 ⁵ cells/mL in a culture medium (DMEM). Take a 30 cm culture plate, place two slides and add 1 ml of culture medium (DMEM), then add 1 ml of the cell suspension of the first group, put it back into the incubator, and incubate for 48 hours. The 30 cm culture dish was taken out, the culture solution (DMEM) was removed, and it was washed with 1 ml of a phosphate buffer solution. Then eight prepared in Example was added at a concentration of 1x10 ⁷ CFU / mL SHB2308 bacterial solution 1 ml, wherein the MOI (Multiplicity of infection, MOI) of 100, at a rate of 1600 rpm, centrifuged for 5 minutes in a cell incubator 20 Minutes, the supernatant was removed and washed three times with 1 ml of phosphate buffer solution, and cultured for 4 hours in a medium containing no fetal bovine serum (DMEM).

Groups 2 and 3 also took the transfected cell lines of Examples 5 and 6, respectively, and co-cultured with SHB2308 solution for 4 hours according to the above steps.

After co-culture for 4 hours, immunofluorescence staining of HCT116 cells of Groups 1 to 3 was performed, respectively, wherein the antibodies used were anti-shigella antibodies and nuclear fluorescent stains (DAPI). The cells of each group were observed at different excitation wavelengths by an inverted conjugate focus microscope, and the results are shown in Annexes 4 to 6.

Annexes 4 to 6 are the results of groups 1 to 3, respectively. In detail, the A map of each annex is the cell type observed in the bright field of view; the B picture of each annex is the nucleus The result of fluorescent staining of fluorescent staining agent (DAPI), the blue part is the nucleus in each figure; the C picture of each accessory is the result of immunofluorescence staining of anti-Shigella antibody, the green part of each figure The strain SHB2308 was detected; the D map of each accessory is the result of overlapping the fluorescence staining of the B and C pictures of each accessory, and the light blue part indicates that the strain SHB2308 is located in the nucleus; E is an enlarged view of the white box in the A diagram of each of the attachments; The F diagram of each attachment is an enlarged view of the white box in the B diagram of each attachment; the G diagram of each attachment is an enlarged view of the white box in the C diagram of each attachment; the H diagram of each attachment It is an enlarged view of the white box in the D picture of each accessory; wherein the magnification of each of the attachments from E to H is 1200 times.

Observing the maps in Annex IV, strain SHB2308 was detected only outside the cell, and observation of the maps of Annexes 5 and 6 showed that the green strain SHB2308 was present in the cytoplasm and was light blue in the nucleus of each figure. The strain SHB2308 shown was present in the nucleus. In other words, comparing the results of Annexes 4 to 6, it is known that HCT116 cells transfected with pFLAG-CMV2 plastid without IpaB gene or IpaB803 gene, after co-culture with strain SHB2308, strain SHB2308 can only be detected outside the cell membrane. , but failed to enter the cell, as shown in Annex IV; whether it is HCT116 cells transfected with recombinant plastid pFLAG-CMV2-IpaB, or HCT116 cells transfected with recombinant plastid pFLAG-CMV2-IpaB803, co-cultured with strain SHB2308 Afterwards, strain SHB2308 can be found in the cytoplasm or/and nucleus, as shown in Annexes 5 and 6 respectively.

Example 11: Co-cultivation of Escherichia coli strain DH5 αα and HCT116 cell line

The cell lines of the transfected plastids prepared in Examples 4 to 6 were respectively divided into three groups, wherein: the first group was pFLAG-CMV2 transfected HCT116 cell line as a control group; the second group was pFLAG- CMV2-IpaB was transfected into HCT116 cell line; the third group was transfected with pFLAG-CMV2-IpaB803 into HCT116 cell line.

E. coli strain DH5 α prepared at a concentration of 1×10 ⁷ CFU/mL prepared in Example 9 was co-cultured with HCT116 cells of Groups 1 to 3, respectively, as in the co-cultivation step described in Example 10. 4 hours.

After co-cultivation for 4 hours, HCT116 cells from group 1 to group 3 were stained with immunofluorescence staining with nuclear fluorescent staining (DAPI), and the groups were observed at different excitation wavelengths by inverted conjugate focus microscope. The results of the cells are shown in Annexes VII to IX.

Annexes VII to IX are respectively the result maps of Groups 1 to 3, in detail: each of the attachments A is a cell type observed under a bright field of view; The result of fluorescent staining of nuclear fluorescent staining agent (DAPI), the blue part is the nucleus in each figure; the green fluorescent system of the C picture of each accessory is the enhanced green fluorescent protein of E. coli strain DH5 α As a result, in other words, the green part in each figure is detected as E. coli strain DH5 α; the D picture of each accessory is the result of overlapping of the B and C pictures of each of the attachments, among them, blue The overlapping part with green is light blue, indicating that the E. coli strain DH5 α is located in the nucleus; the E map of each attachment is an enlarged view of the white box in the A picture of each attachment; It is an enlarged view of the white box in the B picture of each attachment; the G picture of each attachment is an enlarged view of the white box in the C picture of each attachment; the H picture of each attachment is the attachment of each An enlarged view of the white box in the figure D; wherein, the enlargement of the E to H of each of the attachments Rates were 1200 times.

Observing the maps in Annex VII, E. coli strain DH5 α was detected only outside the cell, and each of the figures in Annexes 8 and 9 was observed, showing that the green E. coli strain DH5 α was present in the cytoplasm, and E. coli strain displayed in light blue part of the nucleus DH5 alpha is present in the nucleus. In other words, comparing the results of Annexes VII to IX, it is known that HCT116 cells transfected with pFLAG-CMV2 plastid without IpaB gene or IpaB803 gene can only be detected outside the cell membrane after incubation with E. coli strain DH5α. To E. coli strain DH5 α, as shown in Annex VII; whether it is HCT116 cells transfected with recombinant plastid pFLAG-CMV2-IpaB, or HCT116 cell line transfected with recombinant plastid pFLAG-CMV2-IpaB803, with large intestine After culture of Bacillus strain DH5 α, E. coli strain DH5 α can be found in the cytoplasm or/and nucleus, as shown in Annex VIII and Annex IX.

As can be seen from the results of the above examples, the method for regulating the entry of macromolecules into cells and the method for regulating the entry of macromolecules into cells by the present invention utilizes IpaB gene or IpaB803 gene to transfect cells and make IpaB gene. Or the IpaB803 gene expresses the IpaB protein or the IpaB803 protein in the cell, whereby the macromolecule such as the strain SHB2308 or the Escherichia coli strain DH5α as described in the example can be introduced into the cell from the outside of the cell, and even into the nucleus. Briefly, the technical content and characteristics of the present invention enable the macromolecule to enter the cytoplasm or/and the nucleus of a eukaryotic cell capable of expressing the IpaB gene or the IpaB gene-derived product, and further apply this. In the field of development such as biomedicine.

The first panel is a map of the recombinant plasmid of pFLAG-CMV2-IpaB.

The second figure is a map of the recombinant plasmid of pFLAG-CMV2-IpaB803.

Annex I is a cell map of pCT-CMV2 transfected HCT116 cells, observed by inverted conjugated focal microscope through immunostaining, wherein A is the cell type observed under bright field; B is nuclear fluorescein The result of fluorescent staining of dye (DAPI); C is the result of fluorescent staining of anti-FLAG antibody; D is the result of fluorescent staining of mitochon fluorescent dye (mito-tracker); The picture shows the results of the overlapping of fluorescent staining from B to D.

Annex II is the cell image of the HCT116 cells transfected with pFLAG-CMV2-IpaB by immunofluorescence and observed by inverted conjugated focal microscope. The A picture is the cell type observed under bright field; the B picture is the cell nucleus. The result of fluorescent staining of fluorescent staining agent (DAPI); the picture C is the result of fluorescent staining of anti-FLAG antibody; D is the result of fluorescent staining of mitochon fluorescent dye (mito-tracker) ; E is the result of overlapping of fluorescent staining from B to D.

Annex III is a cell map of pCT-CMV2-IpaB803 transfected HCT116 cells, observed by inverted conjugated focal microscope through immunostaining, wherein A is the cell type observed under bright field; B is the nucleus The result of fluorescent staining of fluorescent staining agent (DAPI); the picture C is the result of fluorescent staining of anti-FLAG antibody; D is the result of fluorescent staining of mitochon fluorescent dye (mito-tracker) ; E is the result of overlapping of fluorescent staining from B to D.

Annex IV is a cell map observed by inverted conjugated focal microscope after transfecting HCT116 cells with pFLAG-CMV2 and co-cultivating with strain SHB2308 by immunostaining, wherein A is the cell type observed under bright field of view. B is the result of fluorescent staining of nuclear fluorescent staining agent (DAPI); C is the result of fluorescent staining of anti-Shigella antibody; and D is the result of overlapping of fluorescent staining of B to C.

Annex V is a transfected HCT116 cell with pFLAG-CMV2-IpaB and the strain After co-cultivation of SHB2308, immunofluorescence, cell diagram observed by inverted conjugated focal microscope, wherein A is the cell type observed in the bright field; B is the nuclear fluorescent stain (DAPI) fluorescence The result of staining; C is the result of fluorescent staining of anti-Shigella antibody; D is the result of overlapping of fluorescent staining of B to C.

Annex VI is a cell map of inverted conjugated focal microscope observed by transfected HCT116 cells with pFLAG-CMV2-IpaB803 and co-cultured with strain SHB2308 by immunostaining, wherein A is a cell observed under bright field of view. Type B; the result of fluorescent staining of nuclear fluorescent staining agent (DAPI); C is the result of fluorescent staining of anti-Shigella antibody; and D is the result of overlapping of fluorescent staining of B to C.

Annex VII is a cell map observed by inverted inverted conjugated focal microscope after transfecting HCT116 cells with pFLAG-CMV2 and co-cultivating with E. coli strain DH5α by immunostaining, wherein A is observed under bright field of view. Cell type; B picture is the result of fluorescent staining of nuclear fluorescent staining agent (DAPI); C picture is the result of detecting enhanced green fluorescent protein of E. coli strain DH5 α; D picture is B to C The result of overlapping light staining.

Annex VIII is the cell map of pFLAG-CMV2-IpaB transfected HCT116 cells and co-cultured with E. coli strain DH5α, and observed by inverted conjugated focal microscope through immunostaining, wherein A is a bright field of view. Observed cell type; B picture is the result of fluorescent staining of nuclear fluorescent staining agent (DAPI); C picture is the result of detecting enhanced green fluorescent protein of E. coli strain DH5 α; D picture is B to C Figure shows the result of overlapping fluorescent staining.

Annex IX is the cell map of pFLAG-CMV2-IpaB803 transfected with HCT116 cells and co-cultured with E. coli strain DH5α, and observed by inverted conjugated focal microscope through immunostaining, wherein A is a bright field of view. Observed cell type; B picture is the result of fluorescent staining of nuclear fluorescent staining agent (DAPI); C picture is the result of detecting enhanced green fluorescent protein of E. coli strain DH5 α; D picture is B to C Figure shows the result of overlapping fluorescent staining.

<110> National Chung Hsing University

<120> A protein that regulates the entry of macromolecules into cells and a method for regulating the entry of macromolecules into cells

<160> 2

<170> PatentIn version3.3

<210> 1

<211> 1743

<212> DNA

<213> Shigella flexneri

<400> 1

<210> 2

<211> 803

<212> DNA

<213> Shigella flexneri

<400> 2

Claims (7)

A method for introducing a macromolecule into a cell comprises the steps of: A. constructing a recombinant plastid having an exogenous gene for expressing a protein comprising at least a positively charged basic amino acid sequence The amino acid sequence is encoded as KKKL; B. the recombinant plastid is transferred into a eukaryotic cell, and the foreign gene is expressed in the eukaryotic cell; C. selecting a large molecule, and step B The eukaryotic cells are co-cultured; D. the macromolecule enters the cell. The method for introducing a macromolecule into a cell according to the first aspect of the patent application, wherein the exogenous gene in the step A is selected from the group consisting of the IpaB gene and the IpaB gene-derived product. A method for introducing a macromolecule into a cell according to claim 1 or 2, wherein in the step D, the macromolecular system is a bacterium. A method for introducing a macromolecule into a cell according to the third aspect of the patent application, wherein in the step D, the bacterium enters the nucleus of the cell. A method for introducing a macromolecule into a cell according to the third aspect of the patent application, wherein in the step D, the bacterium enters the cytoplasm of the cell. A protein that regulates the entry of macromolecules into a cell, the amino acid sequence of which contains less than a positively charged basic amino acid sequence encoded as KKKL. The protein which regulates the macromolecule into the cell according to the sixth aspect of the patent application is translated from a gene selected from the group consisting of the IpaB gene and the IpaB gene-derived product.