CN111620941B

CN111620941B - DA beta 42 and expression vector thereof, and preparation method and application of DA beta 42

Info

Publication number: CN111620941B
Application number: CN202010468801.6A
Authority: CN
Inventors: 苟兴春; 张瑞三
Original assignee: Xian Medical University
Current assignee: Xian Medical University
Priority date: 2020-05-28
Filing date: 2020-05-28
Publication date: 2023-03-17
Anticipated expiration: 2040-05-28
Also published as: CN111620941A

Abstract

The invention discloses a DA beta 42 for establishing an AD disease model and a recombinant plasmid expression vector thereof, and the amino acid sequence of the DA beta 42 is shown in SEQ.ID.NO. 1. The invention also discloses a preparation method of the DA beta 42, the invention constructs prokaryotic recombination of A beta 42 two-string body after modifying and transforming toxic protein A beta 42 from human AD disease, constructs expression plasmid, can obtain a large amount of two-string body protein capable of inducing nerve cells and experimental animals to generate AD-like toxicity after expression and purification, and has low preparation cost and high activity.

Description

DA beta 42 and expression vector thereof, and preparation method and application of DA beta 42

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to DA beta 42, an expression vector and a preparation method of the DA beta 42, and application of the DA beta 42 in establishing an AD disease model in-vivo and in-vitro research.

Background

Alzheimer's Disease (AD) is a typical neurodegenerative disease, and in all cases of dementia, AD accounts for 70%, and age is a high risk factor for the onset of AD. In recent years, the incidence of AD has also increased dramatically with the increasing trend of the global population towards aging. According to the report of the world Alzheimer disease report 2018, 5000 million dementia patients are reported in 2018 all over the world, so that diagnosis and treatment researches on AD have important significance on the patients, families and society, and the AD is a global medical health problem to be urgently solved.

There are two typical lesions in the brains of AD patients: amyloid plaques formed by aggregation of amyloid protein (a β) and neurofibrillary tangles formed by aggregation of hyperphosphorylated Tau protein. There are always different opinions in the research community about the role that Α β aggregation and Tau hyperphosphorylation play in the development of AD, whether it is high or low, who is first and then. However, from either viewpoint, it cannot be denied that a β plays a key role in the development of AD. It is also apparent from a number of related studies that a β can exert AD-like toxic effects on nerve cells both in vivo and in vitro, such as Tau hyperphosphorylation and oxidative stress, as well as synaptic disorders and neuronal damage. Therefore, the A beta is an important molecule for AD research, can be used for simulating the pathogenesis process of AD both in vivo and in vitro, and provides a good experimental model for AD diagnosis and treatment research.

A beta is a protein existing in human brain, and is a series of peptide fragments with different sizes consisting of 39-43 amino acids. A beta is generated by the precursor protein APP through enzyme digestion, and in pathological conditions, the content of A beta 42 consisting of 42 amino acids in the brain is increased; the A beta 42 has strong self-aggregation capability, and finally, products with different aggregation degrees, such as two clusters, oligomers, fibers, plaques and the like, are formed. Therefore, a β 42 is the main toxic substance, but the extent to which it is aggregated is the most toxic, which is always a controversial issue. The oligomer is considered as the main substance exerting toxicity in previous researches, but the polymers of the last few peptide fragments have the greatest toxicity, and the research world has no answer.

Commercial a β 42 is a chemically synthesized monomer that requires a complex series of manipulations to pretreat before use to obtain a toxic polymer. However, this polymer contains polymers composed of different amounts of monomers, and it is unknown which polymer exerts toxicity. Moreover, the chemical synthesis of A beta is very expensive, and the operation process is complex and unstable.

In our previous studies, we found that the modeling effect of DA β 42 is the best by prokaryotic expression of two-string bodies (dimer a β 42, DA β 42) of a β 42, and inducing AD disease models with equivalent doses of commercial a β 42.

Disclosure of Invention

The invention aims to provide a preparation method of DA beta 42.

The second purpose of the invention is to provide a recombinant expression vector for expressing DA beta 42.

The first technical scheme adopted by the invention is a preparation method of DA beta 42, which is specifically carried out according to the following steps:

step 1, obtaining an Abeta 42cDNA sequence from Genebank, wherein the sequence is shown as SEQ ID No. 2;

step 2, optimizing an Abeta 42cDNA sequence through a prokaryotic codon system, wherein the two ends of the sequence contain restriction enzyme cutting sites of Nco I and Xho I, and the sequence is shown as SEQ ID No. 3;

step 3, cloning the optimized DA beta 42cDNA fragment obtained in the step 2 into a pUC57 vector to obtain a pUC57-DA beta 42 recombinant vector;

step 4, transforming the pUC57-DA beta 42 recombinant vector obtained in the step 3 into DH5 alpha escherichia coli, amplifying the pUC57-DA beta 42 recombinant vector, and successfully transforming the pUC57-DA beta 42 recombinant vector into DH5 alpha escherichia coli of the pUC57-DA beta 42 recombinant vector through cloning and screening;

step 5, amplifying and extracting a pUC57-DA beta 42 recombinant vector from DH5 alpha escherichia coli obtained by screening in the step 4, performing enzyme digestion on the obtained pUC57-DA beta 42 recombinant vector by using restriction enzymes Nco I and Xho I respectively, and recovering a DA beta 42cDNA fragment;

step 6, selecting an expression vector PBV220 containing restriction enzyme Nco I and Xho I sites, carrying out enzyme digestion on the expression vector PBV220 by using the restriction enzyme Nco I and Xho I, and recovering a linear vector;

and 7, respectively connecting the DA beta 42cDNA fragment obtained in the step 5 with the PBV220 linear vector obtained in the step 6 by using T4 ligase, then transforming the connected DNA into escherichia coli BL21-DE3 competent cells, and obtaining the BL21-DE3 cells containing the recombinant expression vector PBV220-DA beta 42 through cloning and screening.

And 8, transforming the recombinant expression vector PBV220-DA beta 42 obtained in the step 7 into escherichia coli BL21-DE3 competent cells, and culturing and inducing at 42 ℃ to obtain the DA beta 42.

The first technical solution adopted by the present invention is further characterized in that,

in the step 5, the enzyme digestion reaction conditions are 1h316h at 37 ℃ and inactivation for 20min at 65 ℃.

In the step 6, the enzyme cutting reaction conditions are 1h316h at 37 ℃ and inactivation for 20min at 65 DEG C

The second technical scheme adopted by the invention provides a recombinant expression vector for expressing DA beta 42, the recombinant expression vector is PBV220-DA beta 42, and the recombinant expression vector PBV220-DA beta 42 contains a sequence shown as SEQ ID No. 3.

The invention has the beneficial effects that: toxic protein A beta 42 from human AD diseases is constructed into prokaryotic recombination of A beta 42 two-string body (DA beta 42) for the first time, an expression vector is constructed, and DA beta 42 capable of inducing and establishing an AD disease model can be obtained in large quantity through expression and purification, and the preparation cost is low and the activity is high.

Drawings

FIG. 1 is a diagram showing the results of the expression and purification of DA β 42 analyzed by SDS-PAGE according to the present invention;

FIG. 2 is a graph showing the results of the inhibition of primary neuronal cell viability by DA β 42 according to the present invention;

FIG. 3 is a graph showing the results of the present invention DA β 42 promoting the increase of phosphorylation level of Tau protein in primary neuronal cells;

FIG. 4 is a graph showing the result of the memory level of the injured mice in the novel object recognition model after injecting DA β 42 into the lateral ventricle of the mice according to the present invention.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

The DA beta 42 is used for establishing an Alzheimer disease model, and the amino acid sequence of the DA beta 42 is shown as SEQ ID No. 1.

Example 1 preparation of DA β 42

Step 1, obtaining a DA beta 42 recombinant expression vector:

step 1.1, obtaining an Abeta 42cDNA sequence from Genebank, wherein the sequence is shown as SEQ ID No. 2.

And step 1.2, optimizing an A beta 42cDNA sequence by a prokaryotic codon system, wherein the prokaryotic optimized sequence of the A beta 42cDNA is shown as SE ID No.3, and both ends of the A beta 42cDNA sequence contain enzyme cutting sites of Nco I and Xho I.

Step 1.3, cloning the optimized DA β 42cDNA fragment obtained in step 1.2 into a pUC57 vector to obtain a pUC57-DA β 42 vector.

Step 1.4, transforming the pUC57-DA β 42 vector obtained in step 1.3 into DH 5. Alpha. E.coli competent cells, amplifying the cells, and extracting the pUC57-DA β 42 vector.

Step 1.5, the pUC57-DA beta 42 vector obtained in the step 1.4 is respectively cut by restriction enzymes Nco I and Xho I, the DA beta 42 fragment is recovered, the cutting reaction conditions are 1h316h at 37 ℃ and inactivated for 20min at 65 ℃.

Step 1.6, selecting an expression vector PBV220 containing restriction enzyme Nco I and Xho I sites, carrying out enzyme digestion on the PBV220 vector by using the restriction enzyme Nco I and Xho I, recovering a linear vector, and carrying out enzyme digestion reaction under the conditions of 37 ℃ of 1h316h and 65 ℃ of inactivation for 20min.

And step 1.7, respectively connecting the DA beta 42cDNA fragment obtained in the step 1.5 with the linear vector obtained in the step 1.6 by using T4 ligase, obtaining the recombinant expression plasmid vector PBV220-DA beta 42 of the DA beta 42 by cloning and screening, wherein the connection reaction condition is room temperature (25 ℃) for 10min, and obtaining the DA beta 42 recombinant expression plasmid vector by cloning and screening.

Step 2, induction expression of DA beta 42:

and 2.1, transforming the recombinant expression vector PBV220-DA beta 42 obtained in the step 1 into a competent cell of the escherichia coli BL21-DE 3.

Step 2.2, selecting the monoclonal strain to 5ml LB culture medium containing Amp (100 mg/L), shaking and culturing overnight at 16322 ℃, taking out 1ml bacterial liquid the next day, and inducing the rest 4ml at 65 ℃ for 4h to induce the target protein expression.

And 2.3, taking out 1ml of the bacterial liquid induced in the step 2.2, and ultrasonically (the ultrasonic power is 300w, the work is stopped for 10 seconds again after 15 seconds), so that the bacteria are cracked and the protein is released.

2.4, identifying the protein condition through SDS-PAGE electrophoresis; the expressed whole mycoprotein is stained by Coomassie brilliant blue R250, and the condition for inducing expression and the positive colony clone are determined.

Step 3, affinity purification of DA beta 42:

and 3.1, removing the supernatant of the bacterial lysate obtained in the step 2.3, and filtering the supernatant by using a 22-micron filter membrane.

Step 3.2, balancing the Ni-NTA column by using 100ml of washing liquid containing 40mmol of imidazole, and loading the filtered supernatant with the flow rate controlled to 20330ml/h;

step 3.3, washing the fusion protein with 100ml of a washing buffer containing 40mmol of imidazole to remove non-specifically bound heteroproteins;

step 3.4, eluting the fusion protein by using 15-25ml of elution buffer containing 200mmol of imidazole.

Step 4, identification of DA beta 42:

the bacteria inducing to express the protein are subjected to ultrasonic disruption of thalli to release target protein, then purified by an affinity chromatography column integrated by Ni ions, the purified protein is stored in a refrigerator at the temperature of 70 ℃ below zero, and the amino acid sequence of the bacteria is proved to be consistent with the expected result by protein sequencing, wherein the amino acid sequence is shown in SEQ.NO. 1. The result of the western blot experiment is shown in fig. 1, and as shown in fig. 1, the molecular weight of a β 42 monomer is 4kd, and the band of the purified protein is 8kd, which indicates that the purified protein is the target protein DA β 42.

Step 5, quantitative determination and purity analysis of DA beta 42

(1) Protein quantification was performed by BCA method;

(2) And (3) after the SDS-PAGE gel is stained in Coomassie brilliant blue liquid for 1h, the SDS-PAGE gel is decolorized by a decolorizing liquid fully until the background is colorless, the purity of the purified protein is analyzed by a thin-layer scanner, and the scanning result shows that the purity of the purified protein is as follows: 95.3 percent.

Example 2: in vitro identification of DA beta 42 function of the invention

1. Culture of mouse cortical neurons

By using CO ₂ Over-inhalation to kill newborn mice, soaking in 75% ethanol for 15s for sterilization, taking out cerebrum, placing in pre-cooled D-Hanks buffer solution, peeling off bilateral cortex under dissecting microscope, placing in another clean glass dish containing pre-cooled 0.01M D-Hanks solution, and cutting cortex into 1mm ³ The fragments of the size were digested with 0.125% pancreatin at 37 ℃ for 10-15 minutes and shaken once. Adding 10% FBS to terminate digestion after digestion, gently blowing and beating for 5-8 times with a pipette, standing for 5-8 minutes, transferring cells and supernatant into a new centrifuge tube, centrifuging at 4 deg.C and 800rpm for 5 minutes, discarding supernatant, resuspending precipitate with DMEM +10% FBS, and countingAt 1.25X 10 ⁵ /cm ² Is inoculated in a flask previously coated with polylysine. Placing at 37 deg.C and 5% CO ₂ After 4-6h of culture in the incubator, the culture medium and suspended cells are removed, and replaced by Neurobasal culture medium of NB/2% B27, and the culture is continued, wherein the culture is carried out on day 1, and the culture solution is replaced after 3 days.

2. DA beta 42 inhibits primary neuronal cell viability

As shown in fig. 2, when physiological saline is used as a blank control, the Α β 42 group is used as an experimental group 1, and the DA β 42 group is used as an experimental group 2, and the CCK-8 kit is used to detect the relative growth viability of neurons after neurons are treated for 24 hours by using the Α β 42 and DA β 42 (both at a concentration of 1.0 μ M), it can be seen that the cell viability of both the Α β 42 group and DA β 42 group is significantly lower than that of the control group, and the viability of the DA β 42 group is the lowest, which indicates that both Α β and DA β 42 are toxic to neurons, and the toxicity of DA β 42 to neurons is greater than that of Α β.

3. DA beta 42 promotes the phosphorylation level of Tau protein in primary neuron cells to be increased

After the cells are treated by the DA beta 42 for 24 hours, the cells are lysed, the protein is extracted, and Western blot detection is carried out, the detection result is shown in figure 3, and as can be seen from figure 3, the DA beta 42 (1.0 muM) can remarkably promote the expression level of p-Tau (S202), and under the concentration, the A beta 42 can not remarkably promote the expression level of p-Tau (S202), which indicates that the capacity of the DA beta 42 for promoting the phosphorylation of Tau protein Ser202 is higher than that of the A beta 42.

Example 3: the invention relates to the identification of DA beta 42-induced mouse memory impairment

The artificial cerebrospinal fluid is used as a control group, the A beta 42 group and the DA beta 42 group are used as test groups, the learning and memory conditions of the mouse are detected by using a new object recognition model after injecting the A beta 42 and the DA beta 42 into the lateral ventricle of the mouse for 1 week, the result is shown in figure 4, and the result shows that compared with the control group, the injection of the A beta 42 (100 pmol) into the lateral ventricle of the mouse has no significant influence on the recognition and memory of the new object of the mouse, and the DA beta 42 (100 pmol) can obviously damage the recognition and memory of the new object of the mouse. The result shows that DA beta 42 has obvious memory impairment capability on mice, and DA beta 42 has stronger memory impairment capability than A beta 42 monomer.

In the research of the invention, the two-string body of the A beta 42 (namely DA beta 42) is expressed by a pronucleus, and the modeling of the expression products and the commercial dosage of the A beta 42 is carried out, so that the modeling effect of the A beta two-string body is the best.

Sequence listing

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Claims

A preparation method of DA beta 42 is characterized by comprising the following steps:

step 1, obtaining an Abeta 42cDNA sequence from Genebank, wherein the sequence is shown as SEQ ID No. 2;

step 2, optimizing the A beta 42cDNA sequence obtained in the step 1 through a prokaryotic codon system, wherein both ends of the sequence contain restriction enzyme cutting sites of Nco I and Xho I, and the prokaryotic optimization sequence of the A beta 42cDNA is shown as SEQ ID No. 3;

step 3, cloning the optimized DA beta 42cDNA fragment obtained in the step 2 into a pUC57 vector to obtain a pUC57-DA beta 42 vector;

step 4, transforming the pUC57-DA beta 42 recombinant vector obtained in the step 3 into DH5 alpha escherichia coli, amplifying the pUC57-DA beta 42 recombinant vector, and successfully transforming the pUC57-DA beta 42 recombinant vector into DH5 alpha escherichia coli of the pUC57-DA beta 42 recombinant vector through cloning and screening;

step 5, amplifying and extracting a pUC57-DA beta 42 recombinant vector from DH5 alpha escherichia coli obtained by screening in the step 4, performing enzyme digestion on the obtained pUC57-DA beta 42 recombinant vector by using restriction enzymes Nco I and Xho I respectively, and recovering a DA beta 42cDNA fragment;

step 6, selecting an expression vector PBV220 containing sites of restriction enzymes Nco I and Xho I, carrying out enzyme digestion on the PBV220 by using the restriction enzymes Nco I and Xho I, and recovering a linear vector;

step 7, respectively connecting the DA beta 42cDNA fragments obtained in the step 5 with the PBV220 linear vector obtained in the step 6 by using T4 ligase, then transforming the connected fragments into escherichia coli BL21-DE3 competent cells, and obtaining BL21-DE3 cells containing the recombinant expression vector PBV220-DA beta 42 through cloning and screening;

and 8, transforming the recombinant expression vector PBV220-DA beta 42 obtained in the step 7 into escherichia coli BL21-DE3 competent cells, and culturing and inducing at 42 ℃ to obtain DA beta 42.
2. The method for preparing DA β 42 as claimed in claim 1, wherein the enzyme digestion reaction condition in step 5 is 1h316h at 37 ℃ and 20min at 65 ℃.
3. The method for preparing DA β 42 as claimed in claim 1, wherein the enzyme digestion reaction condition in step 6 is 37 ℃ 1h316h, and 65 ℃ inactivation is 20min.
4. A recombinant expression vector for expressing DA beta 42 is characterized in that the recombinant expression vector is PBV220-DA beta 42, and the PBV220-DA beta 42 contains a sequence shown as SEQ ID No. 3.