AU2021101036A4 - Application of AP-1 in the preparation of markers for autoimmune liver disease - Google Patents

Abstract

The present invention discloses the application of AP-1 in the preparation of markers for autoimmune liver disease, which belongs to the field of medical testing. The present invention discloses the use of AP-1 as a marker for autoimmune liver disease and a fluorescent quantitative assay kit for detecting autoimmune liver disease, which enables rapid and accurate detection of autoimmune liver disease. By using AP-1 expression in peripheral blood as a molecular marker as proposed in the present invention, non-invasive detection of autoimmune liver disease and non invasive dynamic monitoring can be performed, which has important clinical application value. The kit provided by the present invention has high specificity and high sensitivity, overcoming the problems of traditional detection methods that are prone to false negatives and false positives, which provides a new tool for clinical detection of autoimmune liver disease.

Description

Application of AP-1 in the preparation of markers for autoimmune liver

disease

TECHNICAL FIELD

The present invention relates to the field of medical testing, and in particular to the

application of AP-1 in the preparation of markers for autoimmune liver disease.

BACKGROUND

Liver disease is common in China, with up to more than 200 million patients suffering

from chronic liver disease caused by various etiologies. Of these, approximately 4-5% of

patients develop decompensated cirrhosis, liver failure, liver cancer and other end-stage

liver diseases each year due to critical illness. Therefore, how to early and precisely predict

the evaluation, timely diagnosis and effective intervention during the critical course of

CLD, and optimize the protocol of clinical diagnosis and treatment are the keys to reduce

the critical illness and reduce the case fatality rate. Autoimmune liver disease (ALD) is a

specific group of chronic liver diseases caused by immune dysfunction in the body,

including autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), primary

sclerosing cholangitis (PSC),PSC) as well as the mutually overlapping so-called overlap

syndrome. Currently, the detection methods for autoimmune liver disease mainly have

imaging, serum markers, including alt, AST and liver fiber four items, but all of the above

tests have low sensitivity and specificity defects, which cause a high false-positive and

false negative phenomenon in diagnosis. Therefore, providing a biomarker that can be used

to detect autoimmune liver diseases is an important technical issue to be solved in the field.

Activator protein-i (AP-1) is a nuclear transcription factor that binds to cis regulatory

elements of the human metallothionein Ila gene. The canonical AP-1 complex consists of

two subunits, c-Jun and c-Fos, bound to DNA via a leucine zipper. The c-Jun protein, a

dimer formed by hydrophobic interactions via a leucine zipper, is the most predominant

form of AP-1. Activated AP-1 can activate other transcription factors in HSCs by

expressing JunD, one of the AP-1 dimer elements, and work together with AP-1 to induce

the expression of matrix inhibitor of metalloproteinases-1 (TIMP-1), and enhanced TIMP

1 expression can inhibit ECM degradation and promote ECM deposition. AP-1 has not

been reported in prior art as a biomarker for the detection of autoimmune liver disease

SUMMARY

The purpose of the present invention is to provide the application of AP-1 in the

preparation of autoimmune liver disease markers, so as to solve the problems existing in

the prior art, and to conveniently, quickly and accurately detect autoimmune liver diseases.

To achieve the above purpose, the present invention provides the following scheme:

The invention provides an application of AP-1 in the preparation of markers for

autoimmune liver disease.

A kit for detecting autoimmune liver disease is also provided, while the kit includes a

preparation for detecting the autoimmune liver disease marker AP-1.

Preferably, the kit also comprises a fluorescent labeled probe.

Further, the preparation for detecting the autoimmune liver disease marker AP-1

comprises a primer set for detecting AP-1.

Furthermore, sequences of the primer set for detecting AP-1 is shown in SEQ ID No.1

and SEQ ID No.2.

The kit is a fluorescence quantitative detection kit.

The kit for detecting autoimmune liver diseases further comprises a primer set for

detecting internal reference genes.

Additionally, the primer set for detecting the internal reference gene is a primer set

for detecting GAPDH.

The sequence of the primer set for detecting GAPDH is shown in SEQ ID NO.3 and

SEQ DI NO.4.

The invention also provides an application of reagents to detect the expression of AP

1 gene or any subunit in the preparation of autoimmune liver disease diagnostic products.

Beneficial effects:

The invention discloses an application of AP-1 as a marker for diagnosing

autoimmune liver diseases, and provides a kit for detecting autoimmune liver diseases. The

marker and kit provided by the invention can detect autoimmune liver diseases with high

sensitivity and specificity, avoid the problems of false negative and false positive easily

generated in the traditional detection method, and provide a new tool for clinical detection

of autoimmune liver diseases.

BRIEF DESCRIPTION OF THE FIGURES

In order to illustrate more clearly the technical solutions in the embodiments of the

invention or in the prior art, the following is a brief description of the drawings to be used in the embodiments, it is obvious that the drawings in the following description are only some embodiments of the invention, and other drawings can be obtained on the basis of these drawings without any creative work for a person of ordinary skill in the art.

Fig. 1 shows the difference of IgG expression between normal group and model group

mice.

Fig. 2 is the difference of ALT and AST expression between normal group and model

group mice.

Fig. 3 is the graph showing the change of Thl7 and Treg cell ratio between normal

group and model group. In the figure, flow cytometry results of Thl7 cells are shown in

Group A, flow cytometry results of control group on the left, flow cytometry results of

model group in the middle and proportional comparison histogram on the right. The flow

cytometry results of Treg cells in group A, the flow cytometry results of control group on

the left, the flow cytometry results of model group in the middle and the proportional

comparison histogram on the right.

Fig. 4 is the Western blot strip of AP-1 and an analysis histogram of APl/GAPDH in

the liver of mice in normal group and model group.

Fig. 5 is the Western blot map of C-FOS subunit of AP-1 in liver of normal group and

model group mice.

Fig. 6 is the histogram of the expression of C-FOS subunit of AP-1 in peripheral blood

mononuclear cells of healthy people and patients with autoimmune liver disease.

DESCRIPTION OF THE INVENTION

Various exemplary embodiments of the present invention will now be described in

detail, which should not be regarded as a limitation of the present invention, but rather as

a more detailed description of certain aspects, characteristics and embodiments of the

present invention.

It should be understood that the terms described in the present invention are only for

describing specific embodiments, and are not intended to limit the present invention. In

addition, as for the numerical range in the present invention, it should be understood that

every intermediate value between the upper limit and the lower limit of the range is also

specifically disclosed. Intermediate values within any stated value or stated range and every

smaller range between any other stated value or intermediate values within the stated range

are also included in the present invention. The upper and lower limits of these smaller

ranges can be independently included or excluded from the range.

Unless otherwise stated, all technical and scientific terms used herein have the same

meanings as commonly understood by those skilled in the art to which the present invention

relates. Although the present invention only describes preferred methods and materials,

any methods and materials similar or equivalent to those described herein may be used in

the practice or testing of the present invention. All documents mentioned in this

specification are incorporated by reference to disclose and describe methods and/or

materials related to the documents. In case of conflict with any incorporated documents,

the contents of this specification shall prevail.

Without departing from the scope or spirit of the invention, it is obvious to those

skilled in the art that many modifications and changes can be made to the specific

embodiments of the specification of the invention. Other embodiments derived from the

description of the present invention will be apparent to the skilled person. The specification

and examples of this application are only exemplary.

As used herein, "including", "comprising", "having", "containing", etc. are all open

terms, which means including but not limited to.

Unless otherwise specified, the detection and analysis methods adopted by the

invention are conventional experimental methods in the field.

The materials and reagents used by the present invention are all commercially

available.

The mice used in this invention were healthy male C57BL/6 mice from 4 to 6 weeks

of age, all purchased from the Shanghai Experimental Animal Center. After purchase, they

were kept in the general class room of the Animal Center of the First Affiliated Hospital of

Wenzhou Medical University, given standard feed and tap water to drink freely, and the

formal experiments were started after one week of adaptation.

Example 1

Extraction of mouse allogeneic liver antigen S100 and construction of experimental

autoimmune hepatitis model

1. Experimental approach

1) 6 C57BL/6 mice were randomly selected for the extraction of S100 on day 1 and

day 7 (3 mice each time).

2) The mice were given intraperitoneal injection of 10% chloral hydrate (anesthesia)

at a dose of 0.004ml/g, which were fixed on the operating platform.

3) After the cross laparotomy, expose the abdominal cavity, separate the portal vein,

and use a scalp needle to infuse the portal vein with pre-cooled PBS buffer until the liver

turns white.

4) Cut off the liver, place it in pre-cooled PBS buffer, cut it into pieces with scissors,

and then grind it into a tissue homogenate with a tissue homogenizer.

5) Place the liver tissue homogenate in a centrifuge, centrifuge at 150g for 1 hour to

remove the nuclei, then take the supernatant and place it in an ultracentrifuge, centrifuge

at 1000Og for 1hour to obtain the supernatant, which is S100.

6) Place S100 on a pre-installed 90xlcm Sepharose CL-6B separation column.

According to the 3 peaks of UV absorbance, 3 kinds of eluates can be obtained. Take the

first peak eluate and use Amicon Ltra-15 filter centrifugation to control the eluate protein

concentration at 0.5-2g/L.

7) The final S10 first peak eluate is mixed with the same volume of Freund's

complete adjuvant to form a mixture for the construction of a mouse autoimmune hepatitis

model.

8) Model preparation: Inject 1ml of the mixed solution intraperitoneally on the first

day and the seventh day of the experiment (the first injection is calculated on the first day).

9) Model testing: ELISA method was used to measure the IgG levels of the model

group and the normal group, and the Alt/Ast levels of the model group and the normal

group were tested by the Department of Laboratory Medicine, Affiliated Hospital of

Wenzhou Medical University.

2. Experimental results

The difference of IgG level between model group and normal group is shown in Figure

1, and the difference of Alt/Ast level is shown in Figure 2. It can be seen that compared

with normal mice, the levels of IgG, Alt and Ast in the model group increased significantly.

It proved that the mouse autoimmune hepatitis model was successfully established.

Example 2

Flow cytometry detection of Thl7 and Treg cell ratio

1. Experimental method

1) Adjust the cell density to 2x106 /ml.

2) Take 500OL of single cell suspension from each EP tube, add 24L each of

250xPMA/lonomycin and 250xMonesin/BFA, pipette to mix, and incubate at 37°C and

% C02 for 4-6 hours. Mix the solution once every I to 2 hours.

3) Take one tube at random as the isotype control, and the rest are samples to be tested.

4) Add 1.5tL anti-CD4-FITC to each EP tube (including isotype control), and

incubate for 30 minutes at 4°C in the dark.

5) Add 100L of A solution and incubate at room temperature for 15 minutes.

6) Add Iml PBS buffer, centrifuge at 300g for 5 minutes, discard the supernatant (pour

out the supernatant as much as possible).

7) Add 100pL of solution B, add 1.5tL of anti-IL-17-PE to the sample tube (add the

corresponding isotype control in the same volume to the isotype control tube), mix by

pipetting, and incubate for 30 minutes at 4°C in the dark.

8) Add 1ml PBS buffer, centrifuge at 300g for 5 minutes, discard the supernatant.

9) Resuspend the cells in 0.5ml PBS buffer for immediate testing on the machine, or

add 1% paraformaldehyde and store in the dark at 4°C (test on the machine within 24

hours).

10) Thl7 cell flow cytometry: first use forward scatter (FSC) and side scatter (SSC)

to circle lymphocytes, then use CD4' to circle Th cells, and then pass isotype control to

circle CD4+/IL-17+ The cells of are Thl7 cells.

11) Treg cell flow circle gate:Lymphocytes were first circled by forward scatter (FSC)

and side scatter (SSC), then T cells were circled by CD4', and then T cells with

CD4+/CD25+/FPXP3+ were circled by isotype control, which were Treg cells.

2. Experimental results

The results of flow cytometry are shown in Figure 3. It can be seen that the proportion

of Thl7 cells increased and the proportion of Treg cells decreased in the model mice

compared with the control group, demonstrating that the model mice developed

autoimmune responses.

Example 3

Western blot detection of mouse liver AP-1 protein and its C-FOS subunit expression

1. Experimental method

1) Protein denaturation: Take out 50mg of liver tissue of mice from the control group

and the model group, add 1ml of lysate to fully grind, take the supernatant, determine the

concentration by BCA method to adjust the concentration, add 1/4 volume of 5x Loading

Buffer. Mix by pipetting and denature in a hot 100 °C bath for 10 minutes.

2) Glue preparation: Take out the special glass plate for electrophoresis dried in the

oven in advance, put it on the glue preparation rack, mix the prepared 10% separation glue

evenly, slowly add it into the gap between thick plate and thin plate with pipette gun, flatten

the separation glue with ultrapure water, and after standing for 30 minutes, you can see an

obvious line between ultrapure water and separation glue. Drain the upper layer ofultrapure

water, carefully suck the ultrapure water in the gap with filter paper, and slow down the

prepared 5% concentrated glue. And quickly insert the comb into the concentrated gel (be

careful not to generate bubbles), and let it stand for 20 minutes. After the concentrated gel

solidifies, slowly pull out the comb, put the prepared gel in the electrophoresis tank, and

add 1x electrophoresis solution for later use.

3) Sample adding: After mixing the denatured sample to be tested, take 15uL and

slowly add it to the small hole above the concentrated gel, and add protein marker to the

side hole as a standard control.

4) Electrophoresis: Cover the electrophoresis tank and start electrophoresis at 80V.

After the protein marker separates the bands, adjust the voltage to 120V and continue electrophoresis until the target protein runs out of the bottom of the separation gel to terminate the electrophoresis.

5) Glue cutting and film transfer: Cut PVDF film and filter paper in advance according

to the actual situation, activate PVDF film in methanol for 1-2 minutes before use, open

the film transfer clip with the black side facing down, put Shanghai cotton pad and three

layers of filter paper on the black side to smooth and drive away bubbles; Take out the

glass plate in the electrophoresis tank and open it. According to the size of the target strip

(AP-1 is about 1OOKDa; GAPDH is 37KDa), cut off the adhesive tape, carefully put it on

the filter paper, cover it with PVDF membrane and drive away bubbles; Cover the PVDF

membrane with three layers of filter paper and sponge pad, close the membrane transfer

clamp, fix it in the membrane transfer tank, add precooled 1x membrane transfer solution,

place the membrane transfer tank in ice for 300mA, and set the membrane transfer time

according to the molecular weight (IKDa is about 1 minute for membrane transfer).

6) Sealing: Take out the PVDF membrane with the protein side facing up, put it into

the antibody incubator, add the prepared BSA blocking solution, and seal at room

temperature for at least 1.5 hours.

7) Apply the primary antibody: Pour out the blocking solution, add the corresponding

diluted primary antibody (anti-AP-1 dilution is 1:1000, diluted with the primary antibody

dilution), and incubate overnight in the refrigerator at 4°C.

8) Membrane washing: Recover the blocking solution the next day, add 1xTBST

membrane washing solution, place on a shaker to wash the membrane, repeat 3 times for

minutes each time.

9) Apply the secondary antibody: Pour off the washing solution, add the diluted

secondary antibody (diluted ratio is 1:5000, diluted with fresh BSA blocking solution), and

incubate for 1 hour at room temperature.

10) Membrane washing: Recover the secondary antibody, add 1xTBST membrane

washing solution, place it on a shaker to wash the membrane, repeat 3 times for 10 minutes

each time.

11) Exposure and data analysis: prepare ECL luminescent solution (1:1), carefully add

it on PVDF membrane protein surface, pay attention to avoid bubbles, and expose it on the

machine; The gray value of the target protein band was compared with the gray value of

internal reference (GAPDH) to get the ratio, and each band was repeated 3 times to get the

average value, which was analyzed by histogram.

2. Experimental results

The analysis results of AP-1 electrophoresis band and APl/GADPH histogram of

mice liver in normal group and model group are shown in Fig. 4. Compared with the normal

group, the expression of AP-1 in the liver of the model group increased significantly. C

FOS electrophoresis bands of AP-1 subunit in liver of normal group and model group mice

are shown in Fig. 5, which indicates that the expression of AP-1 in liver of model group

mice is greatly increased. These results prove that the expression of AP-1 is significantly

different between autoimmune hepatitis mice and normal mice, and AP-1 can be used as a

biomarker for detecting autoimmune liver diseases.

Example 4

Preparation and application of AP-1 expression detection kit

1. 1. Criteria for inclusion of subjects

The healthy subjects group consists healthy person over 18 years old, without basic

diseases, regardless of gender, no history of liver diseases, autoimmune diseases and

tumors, no history of taking hepatotoxic drugs, no family history of autoimmune diseases

and liver diseases, no other serious physical diseases, no history of drug abuse, and no

pregnancy or lactation period.

The patients with autoimmune hepatitis were selected according to the simplified

diagnostic criteria of autoimmune hepatitis issued by IAIHG in 2008. The age was over 18

years old, and the gender was not limited. The exclusion conditions were: hearing and

visual impairment; history of mental illness; long-term use of sedatives; history of alcohol

and drug abuse; history of severe bleeding in the past 12 months; complicated with hepatitis

A, hepatitis B, hepatitis C and hepatitis E Hepatitis B and other viral hepatitis; recent

(within 1 month) infection; combined with human immunodeficiency virus infection;

combined with nonalcoholic fatty liver disease, alcoholic hepatitis, hereditary liver disease,

drug-induced hepatitis; previous or current tumor diseases; complicated with hepatic

encephalopathy, intractable ascites, bleeding tendency, hepatorenal syndrome; other

autoimmune diseases.

All subjects were fully communicated and signed informed consent.

2. Preparation of human peripheral blood monocytes

(1) Fresh anticoagulated blood was centrifuged at 3000 rpm for 10 min.

2) The supernatant, i.e., plasma, was aspirated off with a rubber-tipped dropper.

3) The remaining peripheral blood with the plasma removed was transferred to a 15ml

centrifuge tube and diluted by adding PBS (peripheral blood: diluent = 1:2).

4) Add Ficoll to a 50 ml centrifuge tube and slowly add diluted peripheral blood along

the inclined wall of the tube (Ficoll: diluted blood = 1:1).

5) Centrifuge at 20°C, 1800 rpm (500-1100g) for 30min.

6) After centrifugation, the centrifuge tube is now divided into four layers from top to

bottom. The first layer is the plasma layer, the second layer is the annular milky white

lymphocyte layer, the third layer is the clear separation fluid layer, and the fourth layer is

the red blood cell layer.

7) Carefully aspirate the second ringed milky white lymphocyte layer with a pipette

into another 15 ml centrifuge tube, add an equal amount of PBS, and mix by blowing.

8) Centrifuge at 1500 rpm for 10 min. and discard the supernatant.

9) Add 5 ml of PBS and mix well by blowing.

10) Repeat step 8 and discard the supernatant thoroughly to obtain peripheral blood

mononuclear cells.

3. Preparation of the kit

The kit is used to detect the relative expression of AP-1 for clinical examination, and

the expression level of AP-1 in peripheral blood mononuclear cells of patients with liver

diseases is detected by real-time fluorescence quantitative PCR, which is helpful for the

diagnosis of autoimmune liver diseases.

Kit components:

(1) Sample total RNA extraction reagents: TRIZOL, chloroform, ethanol, DEPC

water.

(2) Total RNA reverse transcription reagent: Revert First Strand cDNA Synthesis Kit

from Thermo Fisher.

(3) Real-time fluorescence quantitative PCR reaction reagents: upstream and

downstream standard primers for AP-1 and GAPDH (AP-1 primers were designed

according to the C-FOS subunit of AP-1), SYBR Green (Thermo Fisher). The primer

sequences are shown in Table 1 below.

Table 1

Gene

AP-1 Forward (As shown in SEQ ID NO.1) GGCTTCCCTTGATCTGACT

Reverse (As shown in SEQ ID NO.2) ATGCTCTTGACAGGTTCCA

GAPDH Forward (As shown in SEQ ID NO.3) CTCTCCCTCACGCCATC

Reverse (As shown in SEQ ID NO.4) ACGCACGATTTCCCTCTC

4.How to use the kit

1) RNA extraction

(i) Take 50 mg of human peripheral blood mononuclear cells, add 1 ml of Trizol,

grind/blow well and leave for 5 minutes at room temperature to thoroughly separate the

nucleoprotein complex.

(ii) Add 0.2 ml of chloroform, shake vigorously by hand for 15 seconds, wait for 2

minutes at room temperature and then centrifuge at 4°C and 13000 rpm for 15 minutes, and

the solution is visible as three layers, from top to bottom, the clear aqueous phase

(containing RNA), the middle layer and the red phenol-chloroform phase, respectively.

©Transfer the aqueous phase containing RNA to a clean EP tube, add the same

volume of isopropanol, leave for 10 minutes, centrifuge at 13000 rpm for 10 minutes, and

pour off the supernatant.

lml of 75% ethanol was used to wash the precipitate, mix well with a shaker,

centrifuge at 7500g for 5 minutes, pour off the supernatant, and dry the precipitate;

resuspend the RNA precipitate with DEPC water, blow repeatedly with a gun, and measure

the RNA concentration after standing for 10 minutes.

(2) RNA concentration determination: dilute the extracted RNA 15 times with DEPC

water, add it to the colorimetric cup, use ultrapure water as control, and detect the

absorbance at A260 and A280 in UV spectrophotometer; A260/A280 ratio between

1.8-2.0 can be used for subsequent PCR experiments; RNA concentration=OD at

A260x40ng/[lx dilution multiplicity.

(3) Reverse transcription: Configure the reaction system according to the instructions

of the reverse transcription kit, the reaction system is 25uL, lug of total RNA is taken from

each sample, the reaction conditions of reverse transcription are: 60 minutes at 42°C, 5

minutes at 70°C, the cDNA products are used for the subsequent PCR experiments.

4) Detection kit: The kit provided in this embodiment is adopted.

5) Detection method:

10 UL PCR reaction system was prepared, and the reaction system was shown in

Table 2:

Table 2

Reagent Volume(pl) Ultrapure water 2 Forward primer 0.5 Reverse primer 0.5 cDNA 2 SYBR Green 5 Total volume 10

PCR procedure

Pre-denaturation at 95°C for 20-60 seconds; denaturation at 95°C for 1-15 seconds,

annealing at 60°C for 5-30 seconds, extension at 72°C for 30-60 seconds, 40 cycles;

fluorescence was collected at 60°C, and the PCR reaction was performed on a fluorescent

real-time quantitative PCR instrument using SYBR Green as the fluorescent marker for

relative quantification by the AACT method.

©Analysis and judgment of results:

Set baselines and thresholds:

Result determination: AP-1 expression was calculated by ACT = CtAP-1 - CtGAPDH

analysis, if the ACT value was less than 6.0 and the curve had a significant exponential

growth period, it was positive, otherwise it was negative; negative: no detectable sample

Ct value or ACT value was greater than 6.0.

The results of real-time fluorescence quantitative PCR of AP-1 are shown in Figure

6,the expression of C-FOS subunit was significantly higher in patients compared with

normal subjects, i.e. the expression of AP-1 was significantly higher in patients.

The above embodiments only describe the preferred mode of the invention, but do not

limit the scope of the invention. On the premise of not departing from the design spirit of

the invention, various modifications and improvements made by ordinary technicians in

the field to the technical scheme of the invention shall fall within the protection scope

determined by the claims of the invention.

Claims (10)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An application of AP-1 in the preparation of markers for autoimmune liver disease.

2. The kit for detecting autoimmune liver disease, characterized in that the kit includes a

preparation for detecting the autoimmune liver disease marker AP-1.

3. The kit for detecting autoimmune liver disease according to claim 2 with the following

characteristics: the kit also comprises a fluorescent labeled probe.

4. The kit for detecting autoimmune liver disease according to claim 2 with the following

characteristics: the preparation for detecting the autoimmune liver disease marker AP-1

comprises a primer set for detecting AP-1.

5. The kit for detecting autoimmune liver disease according to claim 4 is characterized in

that sequences of the primer set for detecting AP-1 is shown in SEQ ID No.1 and SEQ ID

No.2.

6. The kit for detecting autoimmune liver diseases according to any one of claims 2-5,

which is characterized in that the kit is a fluorescence quantitative detection kit.

7. The kit for detecting autoimmune liver diseases according to claim 6, which is

characterized in that the kit for detecting autoimmune liver diseases further comprises a

primer set for detecting internal reference genes.

8. The kit for detecting autoimmune liver disease according to claim 7, which is

characterized in that the primer set for detecting the internal reference gene is a primer set

for detecting GAPDH.

9. The kit for detecting autoimmune liver disease according to claim 8, which is

characterized in that the sequence of the primer set for detecting GAPDH is shown in SEQ

ID NO.3 and SEQ DI NO.4.

10. Application of reagents to detect the expression of AP-1 gene or any subunit in the

preparation of autoimmune liver disease diagnostic products.

FIGURES 1/3 2021101036

Figure 1. The difference of IgG expression between normal group and model group mice

Figure 2. The difference of ALT and AST expression between normal group and model group mice