CN112142850A

CN112142850A - Human nerve growth factor-lactoferrin recombinant protein and application

Info

Publication number: CN112142850A
Application number: CN201910567696.9A
Authority: CN
Inventors: 吴彦萍; 郭采平; 张战; 陈红波; 吴樱丹; 冀倩倩; 李云富
Original assignee: Shenzhen Weiguang Biological Products Co Ltd
Current assignee: Shenzhen Weiguang Biological Products Co Ltd
Priority date: 2019-06-27
Filing date: 2019-06-27
Publication date: 2020-12-29

Abstract

The invention discloses a human nerve growth factor-lactoferrin recombinant protein and application thereof, wherein the amino acid sequence of the recombinant protein is shown by SEQ ID No. 2. The human nerve growth factor-lactoferrin recombinant protein is used for preparing the medicine for preventing and treating the Alzheimer disease. The invention uses the method of gene engineering to connect two different protein gene segments and express in the mammalian cell, the obtained fusion protein is close to the natural state, and simultaneously has the biological activity of two proteins, thus being capable of solving the defects of single protein in the prior use.

Description

Human nerve growth factor-lactoferrin recombinant protein and application

Technical Field

The invention relates to a nerve growth factor-lactoferrin recombinant protein and application thereof.

Background

Alzheimer's Disease (AD) is a neurodegenerative Disease, the course of Disease is chronic, the AD is the most common type of senile dementia, the AD is mainly manifested as neuropsychiatric symptoms such as progressive memory disorder, cognitive dysfunction, personality change and language disorder, the health of human is seriously affected, and the number of patients in the whole world is estimated to be doubled to 1.15-1.315 hundred million by 2050. The research on Alzheimer's disease has been carried out for hundreds of years, and the research on related therapeutic drugs has been greatly progressed, however, the therapeutic effect of the known drugs is still very limited, so that the research and the development of the drugs for treating AD have very important significance.

The etiology and pathogenesis of AD have not been elucidated, and in recent years, many studies have pointed out that important factors causing disorders such as cognitive, memory, and speech in AD patients include synaptic dysfunction, neuronal loss, neuroinflammation, and cholinergic neurodegeneration in the basal forebrain.

A large number of researches show that in the disease course of AD, the cholinergic nerve of the basal forebrain is one of the first neurons to degenerate and atrophy, seriously influences learning, memory and the like, and is closely related to cognitive disorder of AD patients. Basal forebrain cholinergic neurons require reliance on neurotrophin-mature Nerve Growth Factor (NGF) to maintain their survival, Nerve branching and cholinergic phenotypes, however studies have shown that the amount of mature NGF is significantly reduced in AD patients. Clinical experimental data show that treatment with NGF can promote significant improvement in cognition in AD patients, revealing that treatment with NGF is effective and feasible for alzheimer's disease.

However, in a natural state, NGF can hardly be transported into the Brain through the Blood-Brain Barrier (BBB), which causes that the application of NGF to treat AD encounters a bottleneck, and at present, methods such as intracerebral intubation and ventricular injection are generally adopted in clinical practice to solve the problem that drugs penetrate the Blood-Brain Barrier, but the administration mode has large side effects and is not beneficial to long-term administration. The research of improving the intracerebral delivery of drugs by a non-invasive administration way has become the focus of the domestic and foreign intracerebral targeted drug delivery research because the research can overcome the trauma and the danger caused by surgical operations, and particularly the research of a microparticle drug delivery system which is transferred into the brain through receptor mediation is the most successful. In recent years, the research of Lactoferrin (Lactoferrin, LF) as a carrier of the brain transport system has received increasing attention. The problem of how to improve NGF transfer into the brain by using a brain transfer vehicle is urgently solved, so that a new method is provided for treating neurodegenerative diseases such as Alzheimer's disease and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a human nerve growth factor-lactoferrin recombinant protein.

The second purpose of the invention is to provide a gene for expressing the human nerve growth factor-lactoferrin recombinant protein.

It is a third object of the present invention to provide an expression vector comprising a gene expressing a human nerve growth factor-lactoferrin recombinant protein.

It is a fourth object of the present invention to provide a recombinant animal cell comprising an expression vector expressing a gene of a human nerve growth factor-lactoferrin recombinant protein.

The fifth purpose of the invention is to provide the application of the human nerve growth factor-lactoferrin recombinant protein.

The technical scheme of the invention is summarized as follows:

a human nerve growth factor-lactoferrin recombinant protein is shown in SEQ ID No. 2.

The gene for expressing the human nerve growth factor-lactoferrin recombinant protein has a nucleotide sequence shown in SEQ ID No. 1.

An expression vector comprising a gene expressing a human nerve growth factor-lactoferrin recombinant protein.

A recombinant animal cell comprising the above expression vector.

The application of the human nerve growth factor-lactoferrin recombinant protein in preparing the medicine for preventing and treating the Alzheimer disease.

The invention has the advantages that:

the invention uses the method of gene engineering to connect two different protein gene segments and express in the mammalian cell, the obtained fusion protein is close to the natural state, and simultaneously has the biological activity of two proteins, thus being capable of solving the defects of single protein in the prior use.

Drawings

FIG. 1 shows a plasmid map of PBD-NGF-LTF constructed by using a PiggyBac Dual promoter plasmid (PiggyBac Dual promoter plasmid, PBD plasmid for short). In FIG. 1, the NGF-LTF gene fragment was inserted into MCS, i.e., the multiple cloning site, to construct an expression plasmid. In the schematic representation of the NGF-LTF gene fragment, rectangle 1 represents nerve growth factor NGF, rectangle 2 represents a DNA linker, and rectangle 3 represents lactoferrin LTF.

FIG. 2 is the agarose gel electrophoresis of expression plasmid PBD-NGF-LTF. Lane 1 is DNA marker, the molecular weight shows 100-; lane 2 sample is 0.5. mu.g PBD-NGF-LTF expression plasmid, and two bright bands can be seen, which are PBD-NGF-LTF plasmids, and are respectively located at positions of 5000-8000bp and positions of 8000bp above because of different structures.

FIG. 3 is a graph showing the results of immunoblotting of the fusion protein. FIG. 3 shows successful expression of NGF-LTF fusion protein, in which FIG. 3A shows the results of detection of NGF expression in the fusion protein using an antibody against NGF, a nerve growth factor, and FIG. 3B shows the results of detection of LTF expression in the fusion protein using an antibody against LTF, lactoferrin, and Lane 1-4, first to fourth lanes from the left in FIGS. 3A and 3B show the samples of the fusion protein.

FIG. 4 shows the successful construction of 293F cells stably expressing NGF-LTF fusion proteins.

Wherein FIG. 4A is a picture of selected 293F cells under a microscope bright field; FIG. 4B shows the expression of green fluorescent protein in 293F cells in the same field as FIG. 4A; FIG. 4C shows the detection of NGF-LTF gene fragments in the genome of 293F cells screened by agarose gel electrophoresis.

FIG. 5 is a graph showing the results of measuring nerve growth factor activity of fusion proteins using PC12 cells, in which FIG. 5A is blank control PC12 cells and FIG. 5B is NGF standard-treated PC12 cells; FIG. 5C shows PC12 cells in the negative control group, and FIG. 5D shows PC12 cells treated with the fusion protein.

FIG. 6 is a graph showing the results of fusion protein uptake by Bend.3 cells.

Wherein FIG. 6A is a Bend.3 cell culture fluid sample, the first lane 1 from the left is a blank control group sample, lane 2 is a negative control group sample, and lane 3-7 is a fusion protein group sample; FIG. 6B is a Bend.3 cell sample, the first lane 1 from the left is a blank control group cell sample, lane 2 is a negative control group cell sample, and lane 3-7 is a fusion protein group cell sample; FIG. 6C is a Bend.3 cell sample of a competitive binding assay, and lanes 1-5 from the left are experimental group samples, cells of the experimental group were treated with different amounts of lactoferrin standards and equal amounts of fusion protein, and lane 6 was a blank control group sample NT, and cells of the blank control group were not treated at all.

Detailed Description

The present invention is further described in detail below with reference to specific examples, which are given only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The quantitative tests in the following examples, unless otherwise specified, were set up in triplicate. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples

PC12 cells (rat adrenal medullary chromaffin tumor differentiated cell line) were purchased from ATCC.

Suspension 293F cells (human embryonic kidney cells) were purchased from Gibco.

Bend.3 cells (immortalized mouse brain microvascular endothelial cell line) were purchased from cell banks of Chinese academy of sciences.

The DMEM medium containing 10% fetal bovine serum in the following examples is a medium obtained by adding fetal bovine serum (Hyclone) to a DMEM medium (Gibco) to a volume concentration of 10% fetal bovine serum.

The 293F cell culture medium containing 6mM L-glutamine in the following examples is a medium obtained by adding 200mM L-glutamine (Gibco) to a Freestyle 293F cell culture medium (Gibco) to a concentration of 6mM glutamine.

In the present application, it is preferred that,

the amino acid sequence of human nerve growth factor-lactoferrin recombinant protein (referred to as fusion protein NGF-LTF) is shown in SEQ ID No. 2.

The nucleotide sequence of the gene for expressing the human nerve growth factor-lactoferrin recombinant protein is shown by SEQ ID No. 1.

Example 1

Construction of expression plasmid PBD-NGF-LTF

And sequentially connecting the NGF gene fragment (SEQ ID No.3), the DNA connecting molecule (SEQ ID No.7) and the LTF gene fragment (SEQ ID No.4) in a pcDNA3.1 vector (Addgene) to obtain the fusion gene fragment containing the three DNA fragments. The fusion gene fragment was inserted into PiggyBac Dual promoter Vector (purchased from Biovector NTCC Collection). And transforming the connecting product, selecting a single clone, identifying by using a PCR (polymerase chain reaction) method, sequencing the obtained positive clone, splicing the DNA sequencing result into a sequence shown as SEQ ID No.1 (sequenced by a company of manufacture), and obtaining the amino acid sequence of the corresponding protein as SEQ ID No.2(Primer 5 software conversion sequence).

Preparing 1% agarose gel, and loading 0.5 mu g of PBD-NGF-LTF plasmid with correct sequencing, wherein the result is shown in figure 2, the first lane 1 from the left is a DNA Marker, the second lane 2 is the PBD-NGF-LTF plasmid, and two bright bands can be seen, wherein the bright bands are both the PBD-NGF-LTF plasmid and are respectively located at positions of 8000bp and 8000bp above 5000-plus due to different structures.

Example 2

Expression and identification of fusion protein NGF-LTF

Expression of the fusion protein NGF-LTF

In the experiment, suspended 293F cells are selected for expression of fusion protein, and a transfection reagent is selected from polyethylene PEI (1mg/ml)

(Sigma), details are as follows:

the 293F cells were suspended and counted, and an appropriate number of cells were selected, added to 20ml of Freestyle 293F cell culture medium containing 6mM L-glutamine in a 125ml shake flask to ensure an inoculation density of about 1X 106 cells/ml, and cultured in a cell culture chamber at 37 ℃ under 140rmp with 5% CO2 for 24 hours.

Counting cells 1 hour before transfection, selecting a proper number of cells, removing old culture medium after centrifugation, adding 10ml of fresh 293F cell culture medium to ensure that the cell density is 2.5 × 106/ml-3 × 106/ml, and culturing in a cell culture box at 37 ℃ under the conditions of 140rmp and 5% CO 2.

0.5ml of Opti-MEM (Gibco) was added with 30. mu.g of PBD-NGF-LTF expression plasmid, and 3. mu.g of PiggyBac Transposase Vector (Biovector NTCC Collection) and gently shaken and mixed to prepare solution A; adding 66 mu l of PEI solution into 0.5ml of Opti-MEM, and gently shaking and uniformly mixing to prepare a solution B; both solutions of A, B were allowed to stand at room temperature for 5 min.

Adding the solution B into the solution A, mixing by gently blowing with a pipette, standing for 15min, adding into the cells of step (2), at 37 deg.C, 140rmp, 5% CO₂After 3 hours of culture, 11ml of fresh 293F cell culture medium was added to continue the culture.

After transfection, the cells express the secretory fusion protein NGF-LTF into cell culture solution, and two batches of cell culture solution can be obtained. During the culture, 5ml of fresh 293F cell culture medium is added every 24h, and after 3 days of culture, the cell culture solution is collected by centrifugation. Fresh 293F cell culture medium was added again to resuspend the cells for culture, and the collection of the second batch of cell culture fluid was started and the two batches of cell culture fluid were mixed for subsequent experiments.

2. Identification of fusion protein NGF-LTF

The experiment selects a protein immunoblotting method for detection, and utilizes a specific antibody to identify the expression condition of the fusion protein NGF-LTF, and the specific contents are as follows:

(1) the cell culture fluid collected in step 1 of this example was put into a 1.5ml centrifuge tube, and 5 XSDS-PAGE loading buffer (50% glycerol, 250mM Tris (pH 8.0), 5% beta-mercaptoethanol, 10% SDS, 0.5% bromophenol blue) was added thereto, followed by heat treatment in a metal bath at 100 ℃ for 5 minutes.

(2) Preparing 10% separation gel and 5% concentrated gel according to the SDS-PAGE gel electrophoresis formula, adding the protein sample obtained by the step (1) treatment, and carrying out electrophoresis.

(3) The proteins were transmembrane coated onto PVDF membrane, blocked, and primary anti-lactoferrin antibody (LTF antibody) and primary anti-nerve growth factor (NGF antibody) were added separately and incubated overnight at 4 ℃.

(4) Adding corresponding secondary antibody solution, incubating for 1h at room temperature, adding ECL luminescence solution, exposing, and taking pictures.

The results of Western blotting are shown in FIG. 3, in which FIG. 3A shows that the expression of NGF protein in the fusion protein is detected by using a primary antibody (NGF antibody) against nerve growth factor, and lanes 1-4, the left hand, are samples of the fusion protein, and the results show that the expression of NGF protein is detected; wherein FIG. 3B shows the detection of lactoferrin expression in the fusion protein using a primary antibody (LTF antibody) against lactoferrin, and the left lanes 1-4 are samples of the fusion protein, which shows the detection of lactoferrin expression, and the immunoblot result confirms the successful expression of NGF-LTF.

Example 3

Construction of 293F cells stably expressing NGF-LTF fusion protein

Cell screening

293F cells successfully transfected with PBD-NGF-LTF plasmids express NGF-LTF fusion proteins and green fluorescent protein at the same time and have puromycin (puromycin) resistance, while cells which are not successfully transfected do not express green fluorescent protein and are subjected to apoptosis under puromycin treatment, so that drug screening by using puromycin is facilitated. After the transfected 293F cells were cultured for 48 hours, fresh 293F cell culture medium was changed and puromycin (Seiko Co.) was added to a puromycin concentration of 2. mu.g/ml, and every two days, fresh 293F cell culture medium was changed and puromycin of the same concentration was added for selection for 7 days in total. After the screening was completed, the medium containing puromycin was centrifuged, and a fresh 293F cell culture medium was added and the mixture was cultured in a cell culture incubator at 37 ℃ under 140rmp and 5% CO2 for a total of 30 days. When the cells express the green fluorescent protein under a fluorescence microscope, the green fluorescence intensity is in positive correlation with the expression quantity of the NGF-LTF fusion protein, namely, the green fluorescent protein expressed by the 293F cells can be judged. And (3) sorting 293F cells with higher green fluorescence intensity by using a flow cytometer, culturing and freezing. 293F cells cultured after flow cytometry screening were observed under a fluorescence microscope and photographed.

FIG. 4A shows the expression of green fluorescent protein in 293F cells under the same field of view, and FIG. 4B shows the expression of green fluorescent protein in 293F cells under the microscope.

Detection of NGF-LTF gene fragments in genome

The genome extraction kit (Takara Shuzo Co., Ltd.) is used for extracting the genome DNA of untransfected 293F cells and the 293F cells which are screened and express NGF-LTF fusion protein, and a plurality of detection primers are designed according to the sequence (SEQ ID No.1) of the NGF-LTF to carry out a fluorescent quantitative PCR experiment. The detection Primer 7(SEQ ID No. 5; SEQ ID No.6) with high specificity is selected according to the data of the melting curve to detect the copy number of the NGF-LTF gene in the genome. Wherein, PBD-NGF-LTF plasmid is used as a positive control template, the genome of the 293F cell which is not transfected is used as a negative control template, and the genome of the 293F cell which expresses the NGF-LTF fusion protein is used as an experimental group template. The fluorescent quantitative PCR experiment product is detected by using 1.5 percent agarose gel electrophoresis.

As shown in FIG. 4C, a fluorescent quantitative PCR experiment was performed using the detection Primer 7 as a Primer, and the product of the fluorescent quantitative PCR experiment was detected by electrophoresis in 1.5% agarose gel. The left lane 1 is a DNA Marker, the templates of the lane 2-lane 8 are PBD-NGF-LTF plasmids with different concentrations, the template of the lane 9 is the genome of untransfected 293F cells, and the templates of the lane 10-11 are the genomes of the selected 293F cells expressing the NGF-LTF fusion protein. The experimental results show that the bright bands in lane 10-11 are close to those in lane 2-8, and the molecular weights are consistent with those of the products obtained by using Primer 7 as a Primer, while no band corresponding to the position of lane 9 appears. The results of the experiments indicate that the NGF-LTF gene fragment has been ligated into the genome of 293F cells selected to express NGF-LTF fusion protein, and thus the NGF-LTF gene can replicate as the cells proliferate, thereby maintaining, and stably expressing NGF-LTF fusion protein in the next generation of cells.

In conclusion, the detection of NGF-LTF gene fragments in the genome proves that 293F cells stably expressing NGF-LTF fusion proteins are obtained by the method through drug screening and flow cell screening.

Example 4

Detection of nerve growth factor Activity of fusion proteins

In the experiment, PC12 cells (rat adrenal medullary pheochromocytoma differentiated cell strain) are selected to detect the activity of nerve growth factors of the fusion protein, and PC12 cells have neuron shape-displaying reaction to the nerve growth factors, namely, the cell can stop splitting after being induced by NGF to grow neurite. The specific contents of the experiment are as follows:

(1) PC12 cells were digested, counted and counted at 3X 10⁴One cell/well is inoculated into 24-well plate, 1ml DMEM medium containing 10% fetal bovine serum is added into each well, and the mixture is placed at 37 ℃ and 5% CO₂The cells are cultured in a cell culture box for 24 hours and are randomly divided into a standard substance group, a fusion protein group, a blank control group and a negative control group.

(2) Different groups need different treatments, 1ml of fresh DMEM medium containing 10% fetal calf serum is added before treatment, and three groups share a blank control group.

Standard substance group:

NGF standard (GeneTex) was diluted to 1. mu.g/ml with 10% fetal bovine serum-containing DMEM medium, added in a gradient of 0.3, 1.0, 2.0, 5.0, 10, 15, 20, 25. mu.l/well, and cultured in a 37 ℃ cell culture chamber for 24 hours.

The fusion protein group:

the supernatant of 293F cells stably expressing NGF-LTF fusion protein was collected, added with sodium acetate (30mmol/L) and 2% human serum albumin, adjusted to pH 5.5, sterile filtered, dispensed into 50ml centrifuge tubes, each tube of about 25ml, freeze-dried to powder, and added with 500. mu.L of DMEM medium containing 10% fetal bovine serum to prepare solution C. Adding the mixture according to the gradient of 5, 10, 20, 40, 60, 80 and 100 mu l/hole, and culturing the mixture in a cell culture box at 37 ℃ for 24 hours.

Negative control group:

the supernatant of untransfected 293F cells was collected, added with sodium acetate (30mmol/L), 2% human serum albumin, adjusted to pH 5.5, sterile filtered, aliquoted into 50ml centrifuge tubes, each tube of about 25ml, freeze-dried to powder, and prepared into solution D by adding 500. mu.L of DMEM medium containing 10% fetal bovine serum. Adding the mixture according to the gradient of 5, 10, 20, 40, 60, 80 and 100 mu l/hole, and culturing the mixture in a cell culture box at 37 ℃ for 24 hours.

Blank control group:

the above three groups share a blank control group which is not treated at all.

(3) The morphology of PC12 cells was observed and recorded by photography.

The experimental results show that the PC12 cells in the blank control group and the negative control group have similar shapes and have no obvious neuronal significant response, while the PC12 cells in the standard group and the fusion protein group have obvious neuronal significant response (all pictures are not provided). Wherein FIG. 5A shows the cell morphology of blank control PC12, which is circular, short spindle or triangular, and some of the two cell poles have short protrusions; FIG. 5B shows the morphology of PC12 cells treated with NGF at a concentration of 10ng/ml in the control group, with marked neuronal phenotype, increased numbers of cell processes, multiple other processes extending, with different length and length, and unequal numbers of processes. FIG. 5C shows the morphology of negative control PC12 cells treated with 80. mu.l of solution D, which is similar to the morphology of blank PC12 cells in FIG. 5A, and which did not exhibit significant neuronal phenotype; FIG. 5D shows the morphology of fusion protein group PC12 cells treated with 80. mu.l of solution C, which was similar to the morphology of PC12 cells in the standard group of FIG. 5B, with a clear neuronal visualization. The experimental result shows that the NGF-LTF fusion protein has the activity of nerve growth factor.

Example 5

Detection of intracerebral drug delivery characteristics of fusion proteins

Bend.3 cells (immortalized mouse brain microvascular endothelial cell strain) are selected as a BBB model (blood brain barrier model) to study the intracerebral drug delivery characteristics of the fusion protein NGF-LTF. The immortalized mouse brain microvascular endothelial cell strain Bend.3 is convenient to culture and operate, can still maintain the property of BBB (blood brain barrier) after multiple passages, and has the characteristic of functional barrier. The specific contents of the experiment are as follows:

1. bend.3 cell uptake fusion protein assay

(1) Bend.3 cells were digested, counted and counted at 1X 10⁵One cell/well is inoculated into 24-well plate, 1ml DMEM medium containing 10% fetal bovine serum is added into each well, and the mixture is placed at 37 ℃ and 5% CO₂The cells were cultured in a cell culture chamber for 24 hours and were randomly divided into a blank control group, a negative control group and a fusion protein group.

(2) Different groups were treated differently, and 1ml of fresh DMEM medium containing 10% fetal bovine serum was added before treatment.

The fusion protein group:

the solution C obtained in example 4 was added in a gradient of 10, 20, 40, 80, 100. mu.l/well and incubated at 37 ℃ for 24 hours in a cell incubator.

Negative control group:

mu.l of the solution D obtained in example 4 was added thereto, and the mixture was incubated at 37 ℃ for 24 hours in a cell incubator.

Blank control group:

no treatment is done.

(3) The cell culture solutions in the blank control group, the negative control group and the fusion protein group were each aspirated at 100. mu.l, and 5 XSDS-PAGE loading buffer was added thereto, followed by metal bath treatment at 100 ℃ for 5 min. Discarding the cell culture solution left in the 24-well plate, adding pre-cooled PBS phosphate buffer, gently washing for 3 times, blotting the liquid, adding 1 xSDS-PAGE loading buffer, scraping cells, and treating with metal bath at 100 deg.C for 5 min.

(4) The detection is carried out by a protein immunoblotting method, and an anti-lactoferrin antibody (LTF antibody) is selected as a primary antibody, and is photographed and recorded after exposure.

The result of protein immunoblotting shows that Bend.3 cells can take up NGF-LTF fusion protein. FIG. 6A shows the fusion protein in Bend.3 cell culture, where the first lane 1 from the left is a blank control sample, lane 2 is a negative control sample, and lane 3-7 is a fusion protein sample, and the amount of fusion protein in the culture shows a gradient increase. FIG. 6B shows that in Bend.3 cell samples, the first lane 1 from the left is a cell sample of a blank control group, lane 2 is a cell sample of a negative control group, and lane 3-7 is a cell sample of a fusion protein group, and the amount of the fusion protein taken up by the Bend.3 cell is increased in a gradient manner as the amount of the fusion protein in the culture solution is increased.

Binding competition assay

(1) Bend.3 cells were digested, counted and counted at 1X 10⁵One cell/well is inoculated into 24-well plate, 1ml DMEM medium containing 10% fetal bovine serum is added into each well, and the mixture is placed at 37 ℃ and 5% CO₂The cells were cultured in a cell culture chamber for 24 hours and randomly divided into a blank control group and an experimental group.

(2) Different groups were treated differently, and 500. mu.l of fresh DMEM medium containing 10% fetal bovine serum was replaced before treatment.

Blank control group: no treatment is done.

Experimental groups: lactoferrin standard (Sigma) was added in different amounts, and the mixture was cultured in a cell culture incubator at 0, 100, 200, 400, and 500. mu.g, respectively. After 24h of culture, the same amount of fusion protein was added to each well and the culture was continued for 24 h.

(3) Discarding the cell culture solution in the 24-well plate, adding precooled PBS phosphate buffer solution, gently washing for 3 times, sucking the liquid, adding 1 xSDS-PAGE loading buffer solution, scraping cells, treating the cells in a metal bath at 100 ℃ for 5min, and harvesting a Bend.3 cell sample. Detecting by using a protein immunoblotting method, selecting an anti-nerve growth factor antibody (NGF antibody) as a primary antibody, photographing and recording after exposure, and performing gray scale analysis by using Image J software.

The results of western blotting and grey scale analysis show that the lactoferrin standard substance can generate competitive binding phenomenon with the fusion protein, namely that the band.3 cell absorbs the fusion protein by depending on the property of lactoferrin LTF in the fusion protein, and the fusion protein has specific absorption. After being treated by different amounts of the lactoferrin standard substance, the uptake capacity of the Bend.3 cells to equivalent fusion protein is different, namely, the uptake capacity of the Bend.3 cells to the fusion protein is reduced in a gradient manner along with the increase of the amount of the lactoferrin standard substance. Grey scale analysis shows that the amount of fusion protein taken up by the Bend.3 cells which are not treated by adding the lactoferrin standard substance is set as 1, and the amount of fusion protein taken up by the Bend.3 cells is 0.51, 0.44, 0.42 and 0.31 along with the increase of the amount of the lactoferrin standard substance.

In conclusion, fig. 6 illustrates that the NGF-LTF fusion protein can be taken up by bend.3 cells, and the property of lactoferrin LTF in the fusion protein is relied on, so that the fusion protein has the property of intracerebral drug delivery, the problem that a single protein NGF is difficult to transport into the brain is well solved, and a novel treatment method can be provided for neurodegenerative diseases such as alzheimer disease.

Sequence listing

<110> Shenzhen City Shenzhen satellite photo-biological products stockings Limited

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tgtgcccctg ggtctgaccc gagatctaat ctctgtgctc tgtgtattgg cgacgagcag 1980

ggtgagaata agtgcgtgcc caacagcaac gagagatact acggctacac tggggctttc 2040

cggtgcctgg ctgagaatgc tggagacgtt gcatttgtga aagatgtcac tgtcttgcag 2100

aacactgatg gaaataacaa tgaggcatgg gctaaggatt tgaagctggc agactttgcg 2160

ctgctgtgcc tcgatggcaa acggaagcct gtgactgagg ctagaagctg ccatcttgcc 2220

atggccccga atcatgccgt ggtgtctcgg atggataagg tggaacgcct gaaacaggtg 2280

ttgctccacc aacaggctaa atttgggaga aatggatctg actgcccgga caagttttgc 2340

ttattccagt ctgaaaccaa aaaccttctg ttcaatgaca acactgagtg tctggccaga 2400

ctccatggca aaacaacata tgaaaaatat ttgggaccac agtatgtcgc aggcattact 2460

aatctgaaaa agtgctcaac ctcccccctc ctggaagcct gtgaattcct caggaagtaa 2520

<210> 2

<211> 839

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

Gln Ala Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val

20 25 30

Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp

35 40 45

Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn

50 55 60

Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn

65 70 75 80

Pro Val Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser

85 90 95

Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly

100 105 110

Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys

115 120 125

Val Leu Ser Arg Lys Ala Val Arg Arg Ala Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Arg Arg Arg Ser Val Gln Trp Cys Ala Val Ser

145 150 155 160

Gln Pro Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys

165 170 175

Val Arg Gly Pro Pro Val Ser Cys Ile Lys Arg Asp Ser Pro Ile Gln

180 185 190

Cys Ile Gln Ala Ile Ala Glu Asn Arg Ala Asp Ala Val Thr Leu Asp

195 200 205

Gly Gly Phe Ile Tyr Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro

210 215 220

Val Ala Ala Glu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr

225 230 235 240

Tyr Ala Val Ala Val Val Lys Lys Gly Gly Ser Phe Gln Leu Asn Glu

245 250 255

Leu Gln Gly Leu Lys Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly

260 265 270

Trp Asn Val Pro Ile Gly Thr Leu Arg Pro Phe Leu Asn Trp Thr Gly

275 280 285

Pro Pro Glu Pro Ile Glu Ala Ala Val Ala Arg Phe Phe Ser Ala Ser

290 295 300

Cys Val Pro Gly Ala Asp Lys Gly Gln Phe Pro Asn Leu Cys Arg Leu

305 310 315 320

Cys Ala Gly Thr Gly Glu Asn Lys Cys Ala Phe Ser Ser Gln Glu Pro

325 330 335

Tyr Phe Ser Tyr Ser Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly

340 345 350

Asp Val Ala Phe Ile Arg Glu Ser Thr Val Phe Glu Asp Leu Ser Asp

355 360 365

Glu Ala Glu Arg Asp Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg

370 375 380

Lys Pro Val Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val Pro Ser

385 390 395 400

His Ala Val Val Ala Arg Ser Val Asn Gly Lys Glu Asp Ala Ile Trp

405 410 415

Asn Leu Leu Arg Gln Ala Gln Glu Lys Phe Gly Lys Asp Lys Ser Pro

420 425 430

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

435 440 445

Lys Asp Ser Ala Ile Gly Phe Ser Arg Val Pro Pro Arg Ile Asp Ser

450 455 460

Gly Leu Tyr Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn Leu Arg

465 470 475 480

Lys Ser Glu Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys

485 490 495

Ala Val Gly Glu Gln Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly Leu

500 505 510

Ser Glu Gly Ser Val Thr Cys Ser Ser Ala Ser Thr Thr Glu Asp Cys

515 520 525

Ile Ala Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly

530 535 540

Gly Tyr Val Tyr Thr Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala

545 550 555 560

Glu Asn Tyr Lys Ser Gln Gln Ser Ser Asp Pro Asp Pro Asn Cys Val

565 570 575

Asp Arg Pro Val Glu Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser

580 585 590

Asp Thr Ser Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser Cys His

595 600 605

Thr Ala Val Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu

610 615 620

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

625 630 635 640

Cys Ala Pro Gly Ser Asp Pro Arg Ser Asn Leu Cys Ala Leu Cys Ile

645 650 655

Gly Asp Glu Gln Gly Glu Asn Lys Cys Val Pro Asn Ser Asn Glu Arg

660 665 670

Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Ala Glu Asn Ala Gly

675 680 685

Asp Val Ala Phe Val Lys Asp Val Thr Val Leu Gln Asn Thr Asp Gly

690 695 700

Asn Asn Asn Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe Ala

705 710 715 720

Leu Leu Cys Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg Ser

725 730 735

Cys His Leu Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met Asp

740 745 750

Lys Val Glu Arg Leu Lys Gln Val Leu Leu His Gln Gln Ala Lys Phe

755 760 765

Gly Arg Asn Gly Ser Asp Cys Pro Asp Lys Phe Cys Leu Phe Gln Ser

770 775 780

Glu Thr Lys Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu Ala Arg

785 790 795 800

Leu His Gly Lys Thr Thr Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val

805 810 815

Ala Gly Ile Thr Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu

820 825 830

Ala Cys Glu Phe Leu Arg Lys

835

<210> 3

<211> 417

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgtccatgt tgttctacac tctgatcaca gcttttctga tcggcataca ggcgtcatca 60

tcccatccca tcttccacag gggcgaattc tcggtgtgtg acagtgtcag cgtgtgggtt 120

ggggataaga ccaccgccac agacatcaag ggcaaggagg tgatggtgtt gggagaggtg 180

aacattaaca acagtgtatt caaacagtac ttttttgaga ccaagtgccg ggacccaaat 240

cccgttgaca gcgggtgccg gggcattgac tcaaagcact ggaactcata ttgtaccacg 300

actcacacct ttgtcaaggc gctgaccatg gatggcaagc aggctgcctg gcggtttatc 360

cggatagata cggcctgtgt gtgtgtgctc agcaggaagg ctgtgagaag agcctga 417

<210> 4

<211> 2076

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggccgtagga ggagtgttca gtggtgcgcc gtatcccaac ccgaggccac aaaatgcttc 60

caatggcaaa ggaatatgag aaaagtgcgt ggccctcctg tcagctgcat aaagagagac 120

tcccccatcc agtgtatcca ggccattgcg gaaaacaggg ccgatgctgt gacccttgat 180

ggtggtttca tatacgaggc aggcctggcc ccctacaaac tgcgacctgt agcggcggaa 240

gtctacggga ccgaaagaca gccacgaact cactattatg ccgtggctgt ggtgaagaag 300

ggcggcagct ttcagctgaa cgaactgcaa ggtctgaagt cctgccacac aggccttcgc 360

aggaccgctg gatggaatgt ccctataggg acacttcgtc cattcttgaa ttggacgggt 420

ccacctgagc ccattgaggc agctgtggcc aggttcttct cagccagctg tgttcccggt 480

gcagataaag gacagttccc caacctgtgt cgcctgtgtg cggggacagg ggaaaacaaa 540

tgtgccttct cctcccagga accgtacttc agctactctg gtgccttcaa gtgtctgaga 600

gacggggctg gagacgtggc ttttatcaga gagagcacag tgtttgagga cctgtcagac 660

gaggctgaaa gggacgagta tgagttactc tgcccagaca acactcggaa gccagtggac 720

aagttcaaag actgccatct ggcccgggtc ccttctcatg ccgttgtggc acgaagtgtg 780

aatggcaagg aggatgccat ctggaatctt ctccgccagg cacaggaaaa gtttggaaag 840

gacaagtcac cgaaattcca gctctttggc tcccctagtg ggcagaaaga tctgctgttc 900

aaggactctg ccattgggtt ttcgagggtg cccccgagga tagattctgg gctgtacctt 960

ggctccggct acttcactgc catccagaac ttgaggaaaa gtgaggagga agtggctgcc 1020

cggcgtgcgc gggtcgtgtg gtgtgcggtg ggcgagcagg agctgcgcaa gtgtaaccag 1080

tggagtggct tgagcgaagg cagcgtgacc tgctcctcgg cctccaccac agaggactgc 1140

atcgccctgg tgctgaaagg agaagctgat gccatgagtt tggatggagg atatgtgtac 1200

actgcaggca aatgtggttt ggtgcctgtc ctggcagaga actacaaatc ccaacaaagc 1260

agtgaccctg atcctaactg tgtggataga cctgtggaag gatatcttgc tgtggcggtg 1320

gttaggagat cagacactag ccttacctgg aactctgtga aaggcaagaa gtcctgccac 1380

accgccgtgg acaggactgc aggctggaat atccccatgg gcctgctctt caaccagacg 1440

ggctcctgca aatttgatga atatttcagt caaagctgtg cccctgggtc tgacccgaga 1500

tctaatctct gtgctctgtg tattggcgac gagcagggtg agaataagtg cgtgcccaac 1560

agcaacgaga gatactacgg ctacactggg gctttccggt gcctggctga gaatgctgga 1620

gacgttgcat ttgtgaaaga tgtcactgtc ttgcagaaca ctgatggaaa taacaatgag 1680

gcatgggcta aggatttgaa gctggcagac tttgcgctgc tgtgcctcga tggcaaacgg 1740

aagcctgtga ctgaggctag aagctgccat cttgccatgg ccccgaatca tgccgtggtg 1800

tctcggatgg ataaggtgga acgcctgaaa caggtgttgc tccaccaaca ggctaaattt 1860

gggagaaatg gatctgactg cccggacaag ttttgcttat tccagtctga aaccaaaaac 1920

cttctgttca atgacaacac tgagtgtctg gccagactcc atggcaaaac aacatatgaa 1980

aaatatttgg gaccacagta tgtcgcaggc attactaatc tgaaaaagtg ctcaacctcc 2040

cccctcctgg aagcctgtga attcctcagg aagtaa 2076

<210> 5

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaccaccgcc acagacat 18

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

accactgaac actcctccta c 21

<210> 7

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggaggcggcg gctccggcgg cggaggatca 30

Claims

1. A human nerve growth factor-lactoferrin recombinant protein is characterized in that the amino acid sequence of the recombinant protein is shown in SEQ ID No. 2.

2. The gene for expressing the human nerve growth factor-lactoferrin recombinant protein is characterized in that the nucleotide sequence of the gene is shown by SEQ ID No. 1.

3. An expression vector comprising the gene for expressing a human nerve growth factor-lactoferrin recombinant protein of claim 2.

4. A recombinant animal cell comprising the expression vector of claim 3.

5. The use of the human nerve growth factor-lactoferrin recombinant protein of claim 1 in the preparation of a medicament for preventing and treating alzheimer's disease.