CN112684172A - Method for detecting serum anti-HMGCR antibody of immune necrotic myopathy - Google Patents
Method for detecting serum anti-HMGCR antibody of immune necrotic myopathy Download PDFInfo
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Abstract
The invention provides a method for detecting an anti-HMGCR antibody of serum of immune necrotizing myopathy, which comprises the steps of firstly obtaining an HMGCR gene, constructing a pSJMY-H-mCherry vector with a red coral fluorescence property by a molecular biology method and a recombinant fluorescence label pSJMY-mCherry vector, transiently transfecting HEK293T cells with the vector, and then carrying out immunoreaction on the HMGCR protein antigen expressed in the transfected cells and the serum to be detected. The invention utilizes the double-color indirect immunofluorescence constructed by the red fluorescent protein gene to detect the anti-HMGCR antibody in the human body, has the characteristic of high sensitivity, has definite guiding significance for early diagnosis and curative effect evaluation of patients, and has remarkable social benefit and huge economic benefit.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for detecting an anti-HMGCR antibody of serum of immune necrotizing myopathy.
Background
Idiopathic Inflammatory Myopathies (IIM) are a group of systemic autoimmune myopathies mainly affecting skeletal muscle, and autoimmune abnormalities are the key to the occurrence and development of IIM. According to the recent diagnostic classification criteria of the european neuromuscular disease center and the american cooperative muscular research group, IIMs can be classified into Polymyositis (PM), Dermatomyositis (DM), immune-mediated necrotizing myositis (IMNM), nonspecific myositis (NSM), and inclusion body myositis (sbm), and myositis autoantibodies are added as one of other laboratory examinations to the diagnostic criteria. This indicates that the myositis autoantibodies are of great significance for diagnosis, typing, treatment and prognosis of IIM. IIM is characterized clinically by muscle weakness near the extremities, characteristic rashes, systemic lesions, and the presence of multiple autoantibodies in the serum, which are closely associated with the clinical phenotype.
The necrotic myopathy is a group of diseases which are characterized by subacute or occult onset, symmetrical proximal myasthenia, increase of serum Creatine Kinase (CK), myogenic damage myoelectric expression, Magnetic Resonance Imaging (MRI) mainly takes muscle edema, muscle pathology indicates a large amount of degeneration, necrosis, new myocytes and less inflammatory cell infiltration, and can be caused by autoimmune, poisoning, tumor, endocrine abnormality and other diseases and is easily confused with muscular dystrophy. Among them, autoimmune necrotizing myopathy (autoimmune necrotizing myopathy) is the only hormone-therapeutically effective necrotizing myopathy.
Statins are the first choice drugs for treating hypercholesterolemia, and are mainly classified into natural compounds (such as lovastatin, simvastatin, pravastatin and mevastatin) and completely synthetic compounds (such as fluvastatin, atorvastatin, cerivastatin, rosuvastatin and pitavastatin) which are the most classical and effective lipid-lowering drugs, and are widely used for treating hyperlipidemia. Generally, the statins are divided into three groups, and the application method has very quick response, so the statins generally have the effects of inhibiting the human body from synthesizing cholesterol and reducing the concentration of triglyceride in blood. Statins, which are hydroxymethylglutaryl-coenzyme a (HMG-CoA) reductase inhibitors, block the intracellular mevalonate metabolic pathway by competitively inhibiting endogenous cholesterol synthesis rate-limiting enzyme (HMG-CoA) reductase, and decrease intracellular cholesterol synthesis, thereby feedback-stimulating increased number and activity of Low Density Lipoprotein (LDL) receptors on the cell membrane surface (mainly hepatocytes), and increased serum cholesterol clearance and decreased levels.
In recent years, statins have been increasingly used in clinical applications. However, muscle side effects caused by statins are one of the main reasons limiting their use. Most patients show statin-related self-limited myopathy with diversified clinical characteristics, and light patients can have symptoms of asymptomatic serum CK increase, heavy muscle feeling, stiffness, weakness, pain, exercise tolerance reduction and the like, but after statins are stopped, the muscle symptoms and the serum CK gradually return to normal in weeks to months; striated muscle lysis occurs in a few patients with severe symptoms requiring hospitalization.
When the patient suffers from autoimmune myopathy, the mild patient can have symptoms of heavy muscle feeling, stiffness, weakness, pain, exercise tolerance decline, labor loss and the like, and the severe patient can have rhabdomyolysis, so that the physical health and life of the patient are seriously affected, and even the life of the patient is threatened. If the diagnosis and treatment are not timely carried out, the disability rate and the fatality rate of the patients are increased. If the above symptoms can be diagnosed accurately in time, it is of great significance for the treatment and even the prognosis recovery of the patient.
In the diagnosis of myopathy, histochemical examination of muscle biopsies, although of decisive diagnostic significance, the detection of autoantibodies is also of crucial importance, and the high relevance of myositis-specific autoantibodies to clinical performance and prognosis has received extensive attention. Autoantibodies associated with myositis fall into two categories: the myositis specific autoantibody and myositis related autoantibody can be used as implementation objects of serological detection methods to make up for the defects of histological detection. In immune-mediated necrotizing myositis patients, detection of specific antibodies can distinguish slow-progressing acquired necrotizing myopathy from muscular dystrophy, allowing targeted effective treatment to be given as early as possible.
Data from 6 research centers in europe indicate that there are 3% to 6% of idiopathic inflammatory myopathy patients who are seropositive for anti-SRP antibodies, while the proportion of domestic patients is up to 13%. The studies showed that serum anti-SRP antibodies were associated with immune necrotizing myopathy, with about 20% of patients being seropositive for anti-SRP antibodies. A series of newly discovered specific antibodies for myositis in recent years are all related to immune necrotizing myopathy and severe dermatomyositis, such as Christopher-spine and Watanabe in immune necrotizing myopathyThe relative molecular mass found in serum was 200X 103And 100X 103And the history of use of statin lipid modifying drugs in these patients is as high as 87%. It was thereafter confirmed that 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) recognizes a relative molecular mass of 100X 103The self antigen of (1). Like anti-Signal Recognition Particle (SRP) antibodies, anti-HMGCR antibodies are also specific antibodies for immune necrotizing myopathy, and are newly discovered immunological markers in recent years. The study of Mammen et al showed that 750 patients with proposed inflammatory myopathy had a positive rate of 6% (45/750) for serum anti-HMGCR antibody, and about 92.31% (24/26) of patients over 50 years old had a history of statin lipid-regulating drugs. Studies have shown that immune necrotizing myopathy patients positive for anti-SRP antibodies progress more rapidly and severely than polymyositis, and patients with an early age of onset have more severe clinical symptoms; the patients with immune necrotizing myopathy with positive anti-HMGCR antibody are older and have less muscular weakness. Statistics by Kassardjian et al show that 1/3 is accounted for by serum anti-HMGCR antibody positivity of immune necrotic myopathy patients, which exceeds 1/4 of serum anti-SRP antibody, suggesting the necessity of serum antibody detection in diagnosis of immune necrotic myopathy.
As a subtype of idiopathic inflammatory myopathy, immune necrotic myopathy can be caused by various factors such as tumor, acute viral infection, statin lipid regulators or autoimmunity, while serum anti-HMGCR antibody is mainly found in immune necrotic myopathy patients caused by statin lipid regulators, but is not found in patients taking statin lipid regulators without muscle damage or self-limiting statin-related myopathy. Therefore, the anti-HMGCR antibody is closely related to a statin lipid-regulating drug and is a specific antibody of the statin lipid-regulating drug-mediated immune necrotic myopathy. The necrotic myopathy mediated by the anti-HMGCR antibody has unique clinical and auxiliary examination characteristics, the disease has the characteristics of symmetrical proximal myasthenia, partial accompanied muscle pain, dysphagia and the like, and the serum Creatine Kinase (CK) level reaches the upper limit of a normal reference value by more than 10 times; myoelectricity shows myogenic damage, muscle MRI shows that muscle edema, muscle tissue biopsy is mainly based on muscle cell necrosis and regeneration, mild inflammatory cell infiltration is accompanied or not accompanied, diffuse or multifocal major histocompatibility complex I (MHC.I) expression of muscle cell membranes is up-regulated to be treated by glucocorticoid combined with immunosuppressant, and the anti-HMGCR antibody can be used for curative effect evaluation and follow-up research.
From the above, it is understood that the detection of antibodies in the serum of a patient is of great significance for the diagnosis of the myopathy. The 'anti-HMGCR/SRP autoantibody' is input into the medical apparatus item in the http:// www.sda.gov.cn of the national drug and food supervision and administration network for searching the in vitro diagnostic reagent, and the result shows that the item of '0' meets the requirement, which means that the corresponding in vitro diagnostic reagent which meets the relevant standard does not exist in China although the clinical requirement exists in China so far. Therefore, the development of the in vitro diagnosis reagent for the autoimmune necrotizing myopathy antibody in China has obvious social benefit and simultaneously contains huge economic benefit.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for detecting an anti-HMGCR antibody of immune necrotizing myopathy, and the detection method has the characteristic of high sensitivity.
The purpose of the invention is realized by adopting the following technical scheme:
a method of detecting an anti-HMGCR antibody to immune necrotizing myopathy comprising the steps of:
s1: obtaining an HMGCR gene;
s2: constructing a pSJMY-mCherry recombinant fluorescent label vector;
s3: combining the HMGCR gene obtained in the step S1 with the recombinant fluorescent label vector obtained in the step S2 to construct a recombinant expression vector pSJMY-H-mCherry;
s4: transfecting the recombinant expression vector pSJMY-H-mCherry obtained in the step S3 with cells, and then carrying out immunoreaction on the HMGCR protein antigen expressed in the transfected cells and the serum to be detected for detecting whether an anti-HMGCR antibody exists in the serum to be detected.
Further, the S1 includes the following steps:
1) extracting total RNA from patient tissues, and carrying out reverse transcription by taking the total RNA as a template to obtain total cDNA;
2) obtaining an HMGCR sequence from NCBI and dividing the HMGCR sequence into a front segment and a rear segment, designing a corresponding forward primer and a reverse primer, and carrying out PCR amplification on the cDNA obtained in the step 1) to obtain a corresponding HMGCR gene;
3) respectively carrying out TA cloning on the front fragment and the rear fragment of the HMGCR gene obtained in the step 2) and a T vector, and transforming the ligation product into a competent cell for transformation to obtain a transformant;
4) the transformant of step 3) is picked up for culture.
Further, the forward primer of the front fragment of the HMGCR sequence of step 2) is GGGGCGATCGCCATGTTGTCAAGACTTTTTCGA, and the reverse primer of the front fragment is TTTGCTGAGGTAGTAGGTTGGTCCACCACCCACCGTTCCTATCTCTATAGATGGCAT; the forward primer of the rear fragment of the HMGCR sequence was TTTCCTCAGCAAGCCTGTTTGCAGA, and the reverse primer of the rear fragment was AAAACGCGTGGCTGTCTTCTTGGTG.
Further, the S2 includes the following steps:
5) carrying out PCR amplification on the mCherry red coral protein gene by using a primer pair;
6) constructing a pSJMY-mCherry recombinant fluorescent label vector by using the mChery red coral protein gene obtained in the step 5) and the vector pS 100010.
Further, the forward primer of the mCherry red coral protein gene in the step 5) is TACGCGGCCGCTCGAGATGGTGAGCAAGGGCGAGG, and the reverse primer is CGCGGCCGGCCGTTTAAACCTACTTGTACAGCTCGTCCATGCC.
Further, the S3 includes the following steps:
7) selecting transformants with correct sequencing of the front fragment and the rear fragment obtained in the step 4) for recombination, respectively cutting and recovering the front fragment and the rear fragment from a T carrier, connecting the front fragment and the rear fragment with the empty carrier pSJMY-mCherry containing mChery red coral protein fluorescence at the C end obtained in the step 6) to obtain pSJMY-H-mChery, and converting the connection product into escherichia coli to obtain the transformants;
8) performing colony PCR amplification on the transformant obtained in the step 7) by using a forward primer of the HMGCR front fragment and a reverse primer of the HMGCR rear fragment to determine a positive colony;
9) carrying out amplification culture and enzyme digestion identification on the positive colonies obtained in the step 8).
Further, the S4 includes the following steps:
10) inoculating HEK293T cells into a culture system, transferring the pSJMY-H-mCherry vector obtained in the step 9) and a transfection reagent into the cells together when the concentration of the cells reaches 80%, and performing vector transfection to obtain the cells containing the recombinant expression vector pSJMY-H-mCherry;
11) further fixing, membrane breaking, coating and sealing the transfection vector cells obtained in the step 10);
12) adding a serum to be tested to the HMGCR protein antibody expressed by the cells obtained in the step 11) to perform immunoreaction detection.
Further, the process of step 12) includes:
12.1) diluting the serum sample in proportion;
12.2) discarding the confining liquid on the cell surface obtained in the step 11), adding the diluted serum obtained in the step 12.1) to obtain a uniformly mixed liquid, and incubating;
12.3) discarding the mixed solution in the step 12.2), adding a Triton-containing PBS washing solution into the mixed solution for washing, then adding a corresponding secondary antibody for incubation, and washing the secondary antibody with the Triton-containing PBS washing solution after the incubation is finished;
12.4) adding phosphate buffer solution to the substance obtained in the step 12.3) for immunofluorescence observation.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for detecting an anti-HMGCR antibody of serum of immune necrotizing myopathy, which comprises the steps of firstly obtaining an HMGCR gene, successfully constructing a pSJMY-H-mCherry vector containing red coral fluorescent protein by a molecular biological method and a recombinant fluorescent label vector, transiently transfecting HEK293T cells with the vector, and then carrying out immunoreaction on an HMGCR protein antigen expressed in the cells, the serum to be detected and a corresponding secondary antibody. The invention utilizes the double-color indirect immunofluorescence constructed by the red fluorescent protein gene to detect the anti-HMGCR antibody in the human body, has the characteristic of high sensitivity, has definite guiding significance for early diagnosis and curative effect evaluation of patients, and has remarkable social benefit and huge economic benefit.
Drawings
FIG. 1 is an agarose gel electrophoresis of the PCR results of the pre-HMGCR fragment and the post-HMGCR fragment of the present invention, wherein FIG. 1A shows the pre-HMGCR fragment, FIG. 1B shows the post-HMGCR fragment, and the arrows indicate the size and position of the amplified pre-HMGCR fragment and post-HMGCR fragment;
FIG. 2 is a diagram showing the results of gene sequencing of the HMGCR pre-fragment and post-fragment of the invention;
FIG. 3 is an agarose gel electrophoresis of the PCR result of the fluorescent protein mCherry gene of red coral according to the present invention, wherein the arrow indicates the size and position of the amplified mCherry gene;
FIG. 4 shows a restriction agarose gel electrophoresis image of the pSJMY-mCherry fluorescent tag vector and the restriction agarose gel of the pSJMY-mCherry fluorescent tag vector, wherein M1/M2: DL15000/DL2000, in which lanes 1 and 3 represent pSJMY-mCherry fluorescent vector, and lanes 2 and 4 represent double-restriction pSJMY-mCherry fluorescent vector;
FIG. 5 is an agarose gel electrophoresis image of the pSJMY-mCherry fluorescent vector and HMGCR double restriction enzyme product of the invention, wherein FIG. 5A is HMGCR double restriction enzyme, and an arrow indicates a gel recovery fragment; in FIG. 5B, the No. 1 lane is pSJMY-mCherry fluorescent vector, the No. 2 lane is pSJMY-mCherry fluorescent vector double enzyme digestion, and the arrow indicates the linear vector for glue recovery;
FIG. 6 is an agarose gel electrophoresis of the PCR results of a single colony of a transformant of pSJMY-H-mCherry according to the invention, wherein 6A shows the result of the first amplification step of mCherry after 1-10 transformants were picked: m: DL2000, N: Negative control, P: Positive control, 1-10 is single colony of pSJMY-H-mCherry transformant; 6B shows the result of the second amplification of HMGCR by transformant No. 5-9 in 6A; m: DL15000, N: Negative control, P: Positive control, 5-9 is a single colony of pSJMY-H-mCherry transformant;
FIG. 7 is a restriction agarose gel electrophoresis of the pSJMY-H-mCherry vector of the invention, wherein M: DL10000, 1-5 are plasmids pSJMY-H-mCherry, single cut pSJMY-H-mCherry, double cut HMGCR, single cut HMGCR, respectively;
FIG. 8 is a result chart of Western Blot identification of HMGCR protein expression of the invention;
FIG. 9 is a fluorescence image of the serum anti-HMGCR antibody detected by pSJMY-H-mcherry of the invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict. Unless otherwise specified, most molecular experiments are performed according to conventional experimental conditions, such as the molecular cloning handbook like Sam Brook, or according to the conditions suggested by the manufacturer's instructions.
Examples
The experimental flow chart for detecting the serum anti-HMGCR antibody of the immune necrotic myopathy is shown in figure 1.
S1: obtaining HMGCR genes
The muscle tissue of the patient with the immune necrotic myopathy is from the national hospital of the province of Henan, and the patient uses the informed sample and signs an informed consent.
1) Extracting total RNA of human muscle tissue and obtaining total cDNA
Taking 0.1-0.2g of fresh human muscle tissue, placing the fresh human muscle tissue in a tissue homogenizer, adding precooled 1mL of Trizol liquid into a glass homogenizer, and quickly homogenizing for 15-30 s in an ice bath to fully grind the tissue; then sucking the cell suspension into another Ep tube with the specification of 1.5mL, and standing for 5min at room temperature; then adding 200 mu L of chloroform, shaking vigorously for 15s, mixing uniformly, and standing at room temperature for 3 min; centrifuging at 4 ℃ at 10000r/min for 15min, dividing the centrifuged sample into three layers, absorbing the uppermost colorless aqueous phase containing RNA and transferring the uppermost colorless aqueous phase into another Ep tube; adding 500 μ L isopropanol into the tube, mixing, standing at room temperature for 10min, centrifuging at 4 deg.C at 10000r/min for 10min to obtain RNA precipitate; discarding the supernatant, washing the RNA precipitate with 1mL of 75% ethanol, and centrifuging at 7500r/min at 4 ℃ for 5 min; removing supernatant, and drying at room temperature for 15 min; to the dried precipitate was added 200. mu.l of DEPC treated water (nuclear-free water) to dissolve the precipitate, and the precipitate was stored at-20 ℃ for further use. And (3) detecting the concentration and purity of the RNA obtained by the steps by using a multifunctional microplate reader. And correcting the zero point with DEPC treated water; diluting the RNA sample with DEPC-treated water; OD260, OD280 values and the ratio of OD260/OD280 were read.
cDNA was synthesized by reverse transcription using PrimeScript RT kit, and reaction solutions were prepared according to the components shown in Table 1.
TABLE 1
Reagent | Amount of the composition used |
5×PrimeScript RT Master Mix I | 2.0μL |
Total RNA | 1μg |
RNase Free ddH2O | Adding to 10 μ L |
Total | 10.0μL |
After the reaction solution is mixed gently and evenly, reverse transcription reaction is carried out, and the conditions are as follows: the reaction was first carried out at 37 ℃ for 15min, then at 85 ℃ for 5s, and left at 4 ℃ to obtain cDNA.
2) Obtaining an HMGCR sequence from NCBI and dividing the HMGCR sequence into a front segment and a rear segment, designing a corresponding forward primer and a reverse primer, and carrying out PCR amplification on the cDNA obtained in the step 1) to obtain a corresponding HMGCR gene;
the information on NCBI related to HMGCR is shown in table 2:
TABLE 2
Genetic information | Transcript | Total length of CDS | Amino acids |
HMGCR | I | 2664bp | 888 |
Searching and downloading HMGCR sequence from NCBI website, the invention divides HMGCR gene into front segment and back segment to amplify separately, the corresponding forward primer and reverse primer are shown in table 3, the amplifying process is sent to Shanghai biological engineering company Limited to synthesize.
TABLE 3
3) Respectively carrying out TA cloning on the front fragment and the rear fragment of the HMGCR gene obtained in the step 2) and a T vector, and transforming the ligation product into a competent cell for transformation to obtain a transformant:
wherein the TA cloning comprises the following steps: firstly, a TA cloned connection system is prepared according to the table 4, and after the preparation of the connection system is finished, a connection solution is placed at 16 ℃ for connection for 30min-1 h.
TABLE 4
Reagent | Amount of the composition used |
T-Vector pMD19(Simple) | 1.0μL |
PCR product | 1-2μL(0.1-0.3pmol) |
RNase Free ddH2O | Adding to 5.0 μ L |
Solution I | 5.0μL |
Total | 10.0μL |
The above TA cloning ligation system was transformed into E.coli DH5a by the following method:
3.1) taking the LB resistant plate containing Amp out and standing at room temperature;
3.2) adding the 10 mu L of TA connecting solution into an Ep tube containing competent cell DH5a, and standing for 30min in an ice bath;
3.3) placing the Ep tube obtained in the step 3.2) in a water bath at 42 ℃ for heat shock for 90s, and then carrying out ice bath for 2 min;
3.4) adding 800 mu L of culture medium into the Ep tube in the step 3.3), uniformly mixing, and carrying out mild water bath at 37 ℃ for 60min to obtain a transformed bacterial liquid;
3.5) taking 200 mu L of the transformed bacterium liquid obtained in the step 3.4) and uniformly coating the transformed bacterium liquid on the LB plate obtained in the step 3.1), standing for 5-10min, carrying out inverted culture on the plate and the LB plate at 37 ℃ overnight, observing a resistant plate in the next morning, and selecting bacterial colonies for subsequent experiments.
4) Selecting the transformant in the step 3) for culturing, extracting plasmids, and carrying out target gene PCR and sequencing identification:
part of the transformant colonies picked from the resistant plates obtained in step 3) was added to LB liquid medium containing Amp and cultured for 16h at 180-.
4.1) extracting plasmid: after the bacterial liquid is cultured, the plasmid is extracted by an alkaline lysis method. The method of plasmid miniextraction (plasmid miniextraction kit purchased from Tiangen Biochemical technology (Beijing) Ltd.) is as follows:
4.10) placing the adsorption column CP3 in a collection tube, adding 500 μ L of equilibrium liquid BL into the adsorption column CP3, centrifuging at 12,000rpm for 1min, discarding the waste liquid in the collection tube, and replacing the adsorption column in the collection tube;
4.11) adding 1-5mL of overnight-cultured bacterial liquid into a centrifuge tube, centrifuging at 12,000rpm for 1min, and removing supernatant;
4.12) adding 250 mu L of solution P1 (containing RNase A) into the centrifuge tube with the bacterial sediment, and fully and uniformly mixing by using a pipette to suspend the bacterial sediment;
4.13) adding 250 mu L of solution P2 into the centrifuge tube in the step 4.12), and gently turning over the solution for 6-8 times to fully and uniformly mix the thalli to obtain clear lysate;
4.14) adding 350 mu L of solution P3 into the centrifuge tube in the step 4.13), immediately and gently turning up and down for 6-8 times, fully mixing, generating white flocculent precipitate, and centrifuging at 12,000rpm for 10 min;
4.15) transfer the supernatant from step 4.14) to adsorption column CP3 (adsorption column into collection tube) with pipette to ensure no pellet was aspirated. Centrifuging at 12,000rpm for 30-60s, pouring out waste liquid in the collecting tube, and placing adsorption column CP3 in the collecting tube;
4.16) adding 500 μ L deproteinized liquid PD into the adsorption column CP3, centrifuging at 12,000rpm for 30-60s, pouring off waste liquid in the collection tube, and replacing the adsorption column CP3 in the collection tube;
4.17) adding 600 μ L of rinsing liquid PW (absolute ethyl alcohol is confirmed to be added) into the adsorption column CP3, centrifuging at 12,000rpm for 30-60s, pouring off waste liquid in the collection tube, and putting the adsorption column CP3 into the collection tube; repeating the steps once;
4.18) the adsorption column CP3 was placed in a collection tube and centrifuged at 12,000rpm for 2min to remove the residual rinse from the adsorption column. It should be noted that the ethanol residue in the rinse solution may affect subsequent enzyme reaction (enzyme digestion, PCR, etc.) experiments, and in order to ensure that downstream experiments are not affected by the residual ethanol, the adsorption column CP3 needs to be uncovered and placed at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material;
4.19) placing the adsorption column CP3 in a clean centrifuge tube, dripping 50-100 μ L elution buffer EB into the middle part of the adsorption membrane, placing for 2min at room temperature, centrifuging at 12,000rpm for 2min, and collecting the plasmid solution in the centrifuge tube.
It should be noted that the volume of elution buffer should not be less than 50. mu.L, and that too small a volume will affect recovery efficiency. The pH of the eluent has a large effect on the elution efficiency. If the sequencing is subsequently performed, ddH is required2O is used as eluent, and the pH value is ensured to be in the range of 7.0-8.5, and the elution efficiency is reduced when the pH value is lower than 7.0. And the DNA product should be stored at-20 ℃ to prevent DNA degradation. To increase the recovery of plasmid, the resulting solution can be re-loaded into the adsorption column, left at room temperature for 2min, centrifuged at 12,000rpm (. about.13,400 Xg) for 2min, and the plasmid solution collected in a centrifuge tube.
Because the plasmid provided by the invention is a large plasmid with the size close to 10kb, the using amount of thalli needs to be increased in the extraction process, 5-10mL of overnight culture bacterial liquid is used, the using amounts of P1, P2 and P3 are increased according to the proportion, an elution buffer EB needs to be preheated in a water bath at 65-70 ℃, and the time can be properly prolonged during adsorption and elution so as to increase the extraction efficiency.
4.2) PCR of target gene: the target gene PCR amplification system and conditions are as follows: table 5 shows the prepared reaction solutions.
TABLE 5
Reagent | Amount of the composition used |
DNA template | Less than 1 mug |
Primer 1(HMGCR front/back fragment forward primer) | 0.5μL |
Primer 2(HMGCR front/back fragment reverse primer) | 0.5 |
2×Taq Plus PCR Mix | 12.5μL |
ddH2O | Adding to 25 μ L |
Total | 25μL |
The PCR conditions were set as follows:
step 1.95 ℃ for 2 min;
step 2.94 ℃,30 s;
3.54-60 ℃ for 30 s;
step 4.72 ℃,30 s-2min30 s;
step 5.Go to 2,30 cycles;
step 6.72 ℃, 5 min;
and 8. End.
After the PCR reaction is finished, 1-3 mul of PCR reaction product is taken out to be electrophoresed in agarose gel, EB is dyed, and gel imaging is carried out for photographing. The results of PCR agarose gel electrophoresis of the HMGCR pre-fragment and the HMGCR post-fragment are shown in FIG. 1, which indicates that the molecular weights of the HMGCR gene pre-fragment and the HMGCR gene post-fragment are correct, and the successful amplification is proved.
4.3) sequencing: mu.L of the PCR reaction product with the correct size was taken out and sent to a sequencing company (Shanghai Bioengineering Co., Ltd.) for sequencing, and the sequencing results are shown as SEQ ID NO.7 and SEQ ID NO. 8. The sequencing alignment result is shown in FIG. 2, and the results of DNA MAN analysis show that the sequences of the front segment and the rear segment of HMGCR are correct, the homology is 100%, and the sequencing result is correct.
S2: constructing a pSJMY-mCherry recombinant fluorescent label vector:
5) carrying out PCR amplification on the mCherry red coral protein gene by using a primer;
primer pairs of the mCherry red coral protein gene are shown in table 6:
TABLE 6
FIG. 3 is an agarose gel electrophoresis image of the PCR result of the mCherry red coral fluorescent protein gene, which shows that the band is clear and the molecular weight of the gene is correct, indicating that the mCherry red coral fluorescent protein gene is successfully obtained.
6) Respectively constructing a pSJMY-mCherry recombinant fluorescent label vector (constructed to the C end of the vector) by the mCherry rhodocoral protein gene obtained in the step 5) and the vector pS100010 (the vector is purchased from origin), and identifying the vector by target gene PCR and restriction enzyme cutting.
6.1) extracting plasmid: the plasmid extraction process was the same as that of step 4.1) in S1.
6.2) PCR of target gene: the target gene PCR amplification system and conditions are as follows: table 6 shows the prepared reaction solutions.
TABLE 6
Reagent | Amount of the composition used |
Plasmid DNA template | Less than 1 mug |
Primer 3 (memory forward primer) | 0.5μL |
Primer 4 (memory reverse primer) | 0.5 |
2×Taq Plus PCR Mix | 12.5μL |
ddH2O | Adding to 25 μ L |
Total | 25μL |
The PCR conditions were set as follows:
step 1.95 ℃ for 2 min;
step 2.94 ℃,30 s;
step 3.56 ℃,30 s;
step 4.72 ℃,40 s;
step 5.Go to 2,30 cycles;
step 6.72 ℃, 5 min;
and 8. End.
After the PCR reaction is finished, 1-3 μ L of PCR reaction product is taken to be subjected to an electrophoresis experiment in agarose gel, EB staining and gel imaging photographing are carried out, the result is shown in figure 4, lanes 1 and 3 in the figure show the pSJMY-mCherry fluorescent vector, the band is clear, and the pSJMY-mCherry fluorescent vector is successfully constructed.
6.3) restriction enzyme cleavage: an enzyme digestion reaction system is established according to the table 7, and the prepared enzyme digestion mixture is put into water bath at 37 ℃ for 1-2 h. After the completion, the agarose gel electrophoresis experiment is directly carried out, and staining and gel imaging photographing are carried out by adopting EB.
TABLE 7
Composition of enzyme digestion system | Amount of the composition used |
Plasmid vector | 3μg |
10×Fast | 2μL |
Endonuclease | |
1/XhoI | 0.5 |
Endonuclease | |
2/PmeI | 0.5μL |
ddH2O | Make up to 20. mu.L |
Total | 20μL |
The double digestion results of the vector are shown in lanes 2 and 4 in FIG. 4, and it can be seen from the figure that the fluorescent gene fragment can be clearly seen, and the sizes of the vector and the gene fragment are both correct.
S3: combining the HMGCR gene obtained in the step S1 with the recombinant fluorescent label vector obtained in the step S2 to construct a recombinant expression vector pSJMY-H-mCherry:
7) selecting transformants with correct sequencing of the front fragment and the rear fragment obtained in the step 4) for recombination, respectively cutting and recovering the front fragment and the rear fragment from a T vector, connecting the transformants with correct sequencing of the front fragment and the rear fragment with the empty vector pSJMY-mCherry containing mChery red coral protein fluorescence at the C end obtained in the step 6) to obtain pSJMY-H-mChery, and transforming the ligation product into escherichia coli to obtain the transformants:
7.1) cleavage of the target Gene HMGCR
The enzyme digestion system is shown in Table 8, and the prepared enzyme digestion mixture is put in water bath at 37 ℃ for 1-2 h.
TABLE 8
7.2) cutting the gel to recover and purify the HMGCR fragment:
the gel recovery procedure was performed according to the instructions of the Omega gel recovery kit.
7.21) after the enzyme digestion reaction, directly carrying out agarose gel electrophoresis gel running, when the DNA containing the HMGCR fragment is completely separated, transferring the gel to an ultraviolet lamp to cut the target DNA fragment (as shown in figure 5A), and removing the redundant gel as much as possible.
7.22) transferring the gel block with the target gene obtained in the step 7.21) into a 1.5mL centrifuge tube, and weighing to obtain the weight of the gel block. Approximately determine its volume (assuming its density is 1g/mL), calculate the volume to be 0.2 mL; adding Binding Buffer with the same gel volume, putting the mixture into a water bath at the temperature of 55-65 ℃ for warm bath for 7min until the gel is completely melted, and shaking the mixture to mix uniformly every 2-3 min; it should be noted that after the gel was completely dissolved, the pH of the gel-Binding Buffer mixture was noted. If the pH is more than 8, the yield of DNA is greatly reduced. The mixture was observed for color and if orange or red, 5 μ L of sodium acetate 5.2 at 5M, pH was added to lower the pH. With this adjustment, the color of the mixture will return to the normal pale yellow color.
7.23) transfer 700. mu.L of the DNA-agarose solution obtained in step 7.22) to a Hibind DNA column and pack the column in a clean 2mL collection tube. Centrifuge at 10,000 Xg for 1min at room temperature and discard the filtrate. One of the HiBind DNA columns can hold up to 700. mu.L of solution, and if the volume of the DNA-agarose mixture is greater than 700. mu.L, 700. mu.L of solution can be transferred first, and after centrifugation, the remaining solution is added to the column. However, each HiBind DNA column can bind 25. mu.g to-30. mu.g DNA at most. If a larger yield is to be expected, the samples will need to be loaded separately into the appropriate number of columns.
7.24) the column is replaced in the collection tube, 700. mu.L of SPW Wash buffer to Hibind DNA column, centrifuged at 10,000 Xg for 1min at room temperature, and the filtrate is discarded. Wherein the SPW Wash buffer needs to be diluted by absolute ethyl alcohol according to the requirements of a bottle label before use.
7.25) the column is re-inserted back into the collection tube, and the 7.24) step is repeated twice.
7.26) empty columns were re-inserted back into the collection tube and centrifuged at 10,000 Xg for 1min to dry off the remaining liquid from the column matrix. This step is critical to obtain high yields of DNA.
7.27) the column was placed in a clean 1.5mL centrifuge tube, 30-50 μ L of eluent or sterile water was added to the column membrane and centrifuged at 10,000 Xg for 1min at room temperature, and the solution in the centrifuge tube, i.e., the purified DNA product-HMGCR recovered fragment, was stored at-20 ℃ before use.
7.3) preparation of the purified linearization vector pSJMY-mCherry:
7.31) double enzyme digestion of empty vector pSJMY-mCherry to obtain linearized vector
The enzyme digestion system and the enzyme digestion process refer to 7.1), and the plasmid is replaced by pSJMY-mCherry.
7.32) recovery and purification of the cut rubber
In order to reduce the influence caused by the non-linear vector, directly performing gel running to recover the linear vector after enzyme digestion is finished, wherein the steps are the same as 7.1), and the empty vector enzyme digestion recovery fragment shown in FIG. 5B is the purified linear pSJMY-mCherry vector.
7.4) connection:
after cutting and recovering the gel, connecting the purified HMGCR recovered fragment and the empty vector pSJMY-mCherry enzyme-cut recovered fragment at 22 ℃ for 20-24H by using T4 ligase to obtain pSJMY-H-mCherry. The ligation system is shown in table 9, wherein ligase is purchased from ThermoFisher, and the molar ratio of the HMGCR fragment to the empty vector is 3: 1-10: 1:
TABLE 9
Reagent composition | Amount of the composition used | |
| 1μL | |
10 |
2μL | |
HMGCR recovery fragments | 0.09-0.3pmol | |
Enzyme digestion of empty vector to recover fragment | 0.3pmol | |
ddH2O | Make up to 20. mu.L | |
Total | 20μL |
7.5) transformation:
the process of transforming the escherichia coli is the same as the step 3), and a transformant containing the recombinant expression vector pSJMY-H-mCherry is obtained.
8) Performing colony PCR amplification on the transformant obtained in the step 7) by using a forward primer of the HMGCR pre-fragment and a reverse primer of the HMGCR post-fragment to screen positive clones:
screening positive clones of the transformant in the step 7) by adopting a colony PCR method, selecting a single colony for colony PCR verification, wherein reaction liquid of a PCR amplification system is shown in tables 10-1 and 10-2.
TABLE 10-1
Reagent | Amount of the composition used |
Transformant DNA template | Less than 1 mug |
Primer 1(mcherry forward primer) | 0.5μL |
Primer 2(mcherry reverse primer) | 0.5 |
2×Taq Plus PCR Mix | 12.5μL |
ddH2O | Adding to 25 μ L |
Total | 25μL |
The PCR conditions were set as follows:
step 1.95 ℃ for 2 min;
step 2.94 ℃,30 s;
step 3.56 ℃,30 s;
step 4.72 ℃,40 s;
step 5.Go to 2,30 cycles;
step 6.72 ℃, 5 min;
and 8. End.
After the PCR reaction is finished, taking 1-3 microliter of PCR reaction product to carry out electrophoresis experiment in agarose gel, EB staining and taking pictures by gel imaging. The results are shown in FIG. 6A, in which transformants No. 7-9 among transformants No. 1-10 were seen to amplify the mcherry gene. The HMGCR gene was further amplified using transformant Nos. 5 to 9 as a template, and transformants 7 to 9 were confirmed.
TABLE 10-2
Reagent | Amount of the composition used |
DNA template | Less than 1 mug |
Primer 1(HMGCR front fragment forward primer) | 0.5μL |
Primer 2(HMGCR rear fragment reverse primer) | 0.5 |
2×Taq Plus PCR Mix | 12.5μL |
ddH2O | Adding to 25 μ L |
Total | 25μL |
The PCR conditions were set as follows:
step 1.95 ℃ for 2 min;
step 2.94 ℃,30 s;
step 3.56 ℃,30 s;
step 4.72 ℃,3 min;
step 5.Go to 2,30 cycles;
step 6.72 ℃, 5 min;
and 8. End.
After the PCR reaction is finished, 1-3 mul of PCR reaction products are taken out to be electrophoresed in agarose gel, and EB staining and gel imaging photographing are carried out. The result is shown in FIG. 6B, the PCR result agarose gel electrophoresis of the single clone colony of the pSJMY-H-mCherry transformant shows that the bands of the lanes 7-9 are clear, the forward primer of the front segment and the reverse primer of the rear segment can be used as a pair of primers to amplify the band, the size and the position of the band are both correct, and the fact that the full-length segment of the HMGCR is successfully obtained is proved, namely the corresponding transformant colony is a positive clone.
9) And (3) carrying out enlarged culture on the colonies of the positive clones confirmed in the step 8), namely inoculating the colonies of the positive clones into an LB (Luria Bertani) resistant liquid culture medium containing Amp, and culturing overnight in an air shaker at 37 ℃ at 180-200 rpm. Extracting plasmid, and identifying the recombinant expression vector pSJMY-H-mCherry by means of restriction enzyme cutting.
The enzyme digestion system is shown in Table 11, and after preparation, the enzyme digestion mixture is placed in a water bath at 37 ℃ for 1-2 h.
TABLE 11
And (3) carrying out agarose gel electrophoresis identification on the product after the enzyme digestion is finished, wherein the size of the single digestion (endonuclease SgfI or XhoI) of the enzyme digestion result carrier, the double digestion (endonuclease SgfI-PmeI) of the fusion protein total gene fragment and the double digestion (endonuclease SgfI-MluI) of the HMGCR gene fragment is correct as shown in a figure 7. Restriction enzyme cutting results of colony PCR and the recombinant expression vector verify that the recombinant expression vector pSJMY-H-mCherry is successfully constructed.
S4: transfecting the recombinant expression vector pSJMY-H-mCherry obtained in the step S3 with cells, and then carrying out immunoreaction on an HMGCR protein antigen expressed in the transfected cells and serum to be detected for detecting whether anti-HMGCR antibodies exist in the serum to be detected:
10) inoculating HEK293T cells into a 24-hole culture plate and a chamber glass slide system, and when the cells reach 80% concentration after 24 hours, respectively mixing the pSJMY-H-mCherry vector obtained in the step 9) with a transfection reagent (Turbo 8.0) according to the mass-volume ratio of 0.1 mu g: mu.L of the vector was transferred into HEK293T cells for transfection. Is discharged to CO2And after culturing for 48 hours in the incubator, observing the transfection efficiency under a fluorescence microscope, recording and photographing, and carrying out subsequent experiments under the condition that red or green fluorescence exists and the transfection efficiency reaches more than 60-80%.
11) The supernatant from the culture medium of HEK293T cells transfected in step 10) was removed first, the transfected cells were washed with PBS, the washing solution PBS was discarded, and then the cells were fixed with paraformaldehyde for 20min and washed 2 times with PBS. Then adding Triton reagent (brand: Sigma) and BSA reagent (brand: GENVIEW) for membrane rupture and blocking for 3 hours to overnight to obtain permeable recombinant expression vector pSJMY-GFPN-cells of-H.
Identification and evaluation of transiently transfected cells: extracting whole protein from transfected cells to carry out protein immunity experiment, and carrying out HMGCR protein expression level verification on the cells containing the recombinant expression vector:
11.1) Total protein extraction:
(1) pouring out the culture solution, and reversely buckling the bottle on the absorbent paper to make the absorbent paper absorb the culture solution, or standing the bottle upright for a while to make the residual culture solution flow to the bottom of the bottle and then sucking away the residual culture solution by using a pipettor;
(2) 3mL of 4 ℃ precooled PBS (0.01M pH 7.2-7.3) was added to each flask of cells, the flask was kept flat, the cells were washed by gentle shaking for 1min, and then the wash solution was discarded. Repeating the operation twice, washing the cells for three times to wash out the culture solution, and placing the culture bottle on ice after the PBS is completely removed;
(3) according to the lysis solution: PMSF (100mM) ratio 1 mL: adding 10 mu L of the mixture into the culture bottle in the step (2), wherein the PMSF can be mixed with the lysate when the PMSF is shaken up until no crystal exists, and placing the culture bottle on ice after shaking up;
(4) adding lysis solution to the culture bottle obtained in the step (3), adding 100-400 mu L of lysis solution containing PMSF to each bottle of cells, and cracking the cells on ice for 30min, wherein the culture bottle needs to be shaken back and forth frequently to fully crack the cells;
(5) after the lysis, the cells are quickly scraped at one side of the culture bottle by a clean scraper, and then cell fragments and lysis solution are moved to a 1.5mL centrifuge tube by a gun, so that the whole operation is carried out on ice as far as possible;
(6) centrifuging the centrifuge tube of step (5) at 12000rpm at 4 ℃ for 5 min.
(7) And (4) subpackaging the centrifuged supernatant obtained in the step (6), transferring the supernatant into a centrifuge tube for 0.5min, and storing the centrifuge tube at the temperature of minus 20 ℃ or minus 70 ℃ for later use.
11.2) preparation of electrophoresis gel SDS-PAGE and protein electrophoresis
(1) Cleaning the glass plate: one hand fastens the glass plate and the other hand dips in washing powder and lightly scrubs. Washing both sides with tap water, washing with distilled water, and air drying;
(2) pouring glue and loading sample: aligning the glass plates cleaned in the step (1), putting the glass plates into a clamp, vertically clamping the glass plates on a frame, and preparing for gluing; when pouring the gel, 1mL of the gel can be sucked by a 1mL gun and discharged along the glass, the solution is slowly added into the assembled plate until the gel height is about 6cm, and the height of 1.5cm is reserved to prepare the concentrated gel. The application method of the concentrated gel comprises the following steps: 4% concentrated gum was used with < 10% separation gum, 6% concentrated gum with > 10% separation gum. And then adding a layer of water on the glue, gelling the glue after liquid sealing is quicker, and standing the glue at room temperature for 0.5-1 h until the polymerization is complete. When there is a line of refraction between the water and the glue, it indicates that the gel has been formed. Stopping for 3min to solidify the glue, pouring off the water on the upper layer of the glue, and sucking the water with absorbent paper.
Filling the rest space with concentrated glue, and inserting a comb into the concentrated glue, wherein the glue flows down along the glass plate during glue filling, and the comb is kept horizontal during comb insertion, so as to avoid bubbles in the glue. Because the volume can shrink when the glue solidifies and make the sample loading volume of the sample loading hole reduce, glue needs to be supplemented in the process of concentrated glue solidification. After the concentrated gel is solidified, the two hands respectively hold the two ends of the comb to vertically and upwards slightly pull out the comb. Gel polymerization is carried out for about 0.5 to 1 hour.
Washing the concentrated gel with water, putting the washed gel into an electrophoresis tank, extracting the protein, mixing the gel with about 10-15 mu L of 5 xSDS loading buffer solution according to the proportion of 5:1, adding the mixture into a 1.5mL centrifuge tube, and boiling the centrifuge tube in boiling water for 3-5 min to completely denature the protein. The electrophoresis process comprises the steps of firstly setting the voltage to be 80V, increasing the voltage to be 120-150V when a sample reaches the junction of the concentrated gel and the separation gel, stopping electrophoresis when bromophenol blue migrates to the bottom of the gel, and then carrying out membrane rotation.
11.3) transfer of films
A sufficient amount of transfer buffer was first prepared to fill the running well, and another 200mL of transfer buffer was prepared for equilibration of the gel and membrane and wetting of the filter paper. Taking out the gel after electrophoresis, and rinsing the gel in a membrane transfer buffer solution for 15-30 min; soaking the filter paper in a transfer buffer solution for 30 s; wetting the PVDF membrane in methanol for 15s to ensure that the membrane becomes semitransparent, soaking the PVDF membrane in double distilled water for 2min, and then putting the PVDF membrane in a transfer buffer solution for balancing for 5 min.
The clamp is opened to keep the black side horizontal, a sponge pad is arranged on the black side, and a glass rod is repeatedly squeezed to remove air bubbles in the sponge pad. Before stripping the glue, the glass plate is firstly removed, the concentrated glue is lightly scraped, the separation glue is prevented from being scraped to be broken, and meanwhile, the mark is made. The separation gel was carefully overlaid on the filter paper, and the air bubbles were similarly expelled by glass rod extrusion. PVCF membranes were then covered on the separation gel to exclude air bubbles. Three filter papers were then overlaid on the PVDF membrane and the air bubbles were removed. And finally covering the spongy cushion, and closing the clamp after removing the bubbles. The whole operation process is carried out in the transfer liquid, and air bubbles need to be continuously removed. And placing the clamp into the transfer groove, wherein the black surface of the clamp is opposite to the black surface of the transfer groove, and the red surface of the clamp is opposite to the red surface of the transfer groove. The film transfer apparatus was placed on ice at 200mA for 2 h.
11.4) sealing
The membrane obtained in step 11.3) is transferred to a dish containing the blocking solution and blocked with the blocking solution at room temperature for 2h or overnight at 4 ℃ with shaking on a decolourization shaker.
11.5) Primary antibody
Diluting a primary antibody (brand: Santacuz/Affinity) with a blocking solution to be remarked between 1:4000 and 1:8000, taking out the membrane from the blocking solution obtained in the step 11.4), sucking residual liquid by using filter paper, putting the membrane protein face upwards on an antibody liquid surface, and incubating for 1-2h or overnight at 37 ℃ on a decolorizing shaker.
11.6) washing
Washing with TBST at room temperature for 5min for 3 times, and if the background is higher, the washing time can be increased; if the bands are found to be faint in the preliminary experiment, the primary antibody can be incubated overnight at 4 ℃.
11.7) Secondary antibody
The secondary antibody dilutions were contacted with the membrane and incubated for 2h at 37 ℃. Wherein the secondary antibody brand: beijing Ding Guoshang Biotechnology Limited liability company, diluent brand: shanghai flash crystal molecular Biotechnology, Inc. 11.8) washing
Washing the membrane obtained in the step 11.7) with TBST at room temperature for 5min for 3 times by a decoloring shaker; if the background is found to be higher in the preliminary experiment, the washing time and the washing time can be properly increased; if the bands were found to be faint in the preliminary experiment, the secondary antibody may be incubated overnight at 4 ℃.
11.9) Exposure and photography
Mixing solution A and solution B (ECL ultrasensitive luminescence detection kit purchased from Epizyme Yazyme) in equal volume in a centrifuge tube according to the kit instruction, spreading the solution on membrane protein after 1min, removing residual liquid after 2min of dark room reaction, packaging and placing in an X-ray film clamp. The film was scanned or photographed, a gel image processing system was used and the bands were analyzed.
The results are shown in fig. 8, which shows successful expression of HMGCR protein in cells.
12) Adding a test serum (150 serum samples) to wells of a cell culture plate (NEST) containing the HMGCR protein expressed in the cells obtained in step 11) to perform an immunoreaction:
12.1) taking serum of suspected clinical immune necrotizing myopathy patients as a specimen, and carrying out proportional dilution:
12.2) discarding the sealing liquid on the surface of the transfected cells, adding the diluted serum sample obtained in the step 12.10 to obtain a uniformly mixed liquid, and incubating for 30min-2h at 37 ℃;
12.3) adding triton (brand: sigma) was washed with PBS wash, and then anti-human Alexa was added to cells of the recombinant expression vector pSJMY-H-mcherry in response thereto488 second antibody, incubating for 30min-2h, adding PBS washing solution containing triton after incubation is finished, and washing;
12.4) adding a certain volume of phosphate buffer solution for immunofluorescence observation.
And (3) observing and photographing by using blue excitation light and green excitation light of a fluorescence microscope respectively, and recording the excitation light observation sequence of two different types of fluorescent label carriers. The invention utilizes a two-color indirect immunofluorescence method to determine the condition of the anti-HMGCR antibody in human serum. As shown in FIG. 9, the recombinant expression vector pSJMY-H-mcherry is transfected into corresponding HEK293T cells to express fusion protein of red fluorescent protein and HMGCR protein, and immune reaction occurs after serum is added, if fluorescence of CBA shows red and green fluorescence respectively. Fig. 9A shows that red fluorescence can indicate a vector transfection effect and a corresponding fusion protein expression position, a green fluorescence result shows a red cell morphology, and the red fluorescence cell morphology and the green fluorescence cell morphology position are consistent and can be fused to show a yellow fluorescence effect, which indicates that the serum sample contains an anti-HMGCR antibody, and that the serum sample contains an HMGCR antibody, i.e., the serum to be detected is a positive sample; as a control, fig. 9B shows that the fluorescence of CBA only exhibits red fluorescence but no green fluorescence, the red fluorescence indicates the transfection effect of the vector and the expression position of the corresponding fusion protein, the green fluorescence result has no obvious cell morphology, and the yellow fluorescence effect is not exhibited after fusion, indicating that the serum sample does not contain the anti-HMGCR antibody, i.e., the serum to be detected is a negative sample. The method provided by the invention can detect whether the serum of a suspected patient contains the anti-HMGCR antibody, thereby diagnosing whether the suspected patient has the immune necrotic diseases and assisting the final clinical diagnosis.
In conclusion, the recombinant expression fluorescent vector constructed by the invention has higher sensitivity when being used for detecting the anti-HMGCR antibody in a human body by a two-color indirect immunofluorescence method, has definite guiding significance for early diagnosis and curative effect evaluation of a patient, and has remarkable social benefit and huge economic benefit.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Sequence listing
<110> Zhengzhou university
<120> method for detecting immune necrotizing myopathy serum anti-HMGCR antibody
<130> description
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cgcggccggc cgtttaaacc tacttgtaca gctcgtccat gcc 43
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actggtaaca ataagatctg tggttggaat tatgaatgtc caaagtttga agaggatgtt 180
ttgagcagtg acattataat tctgacaata acacgatgca tagccatcct gtatatttac 240
ttccagttcc agaatttacg tcaacttgga tcaaaatata ttttgggtat tgctggcctt 300
ttcacaattt tctcaagttt tgtattcagt acagttgtca ttcacttctt agacaaagaa 360
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agcacattag caaagtttgc cctcagttcc aactcacagg atgaagtaag ggaaaatatt 480
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gtgattggag ttggtaccat gtcaggggta cgtcagcttg aaattatgtg ctgctttggc 600
tgcatgtcag ttcttgccaa ctacttcgtg ttcatgactt tcttcccagc ttgtgtgtcc 660
ttggtattag agctttctcg ggaaagccgc gagggtcgtc caatttggca gctcagccat 720
tttgcccgag ttttagaaga agaagaaaat aagccgaatc ctgtaactca gagggtcaag 780
atgattatgt ctctaggctt ggttcttgtt catgctcaca gtcgctggat agctgatcct 840
tctcctcaaa acagtacagc agatacttct aaggtttcat taggactgga tgaaaatgtg 900
tccaagagaa ttgaaccaag tgtttccctc tggcagtttt atctctctaa aatgatcagc 960
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ttctttgaac aaacagagac agaatctaca ctctcattaa aaaaccctat cacatctcct 1080
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gaggttataa aacccttagt ggctgaaaca gataccccaa acagagctac atttgtggtt 1260
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acagaaggtt gtcttgtggc cagcaccaat agaggctgca gagcaatagg tcttggtgga 1740
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ataaaggagg catttgacag cactagcaga tttgcacgtc tacagaaact tcatacaagt 1920
atagctggac gcaaccttta tatccgtttc cagtccaggt caggggatgc catggggatg 1980
aacatgattt caaagggtac agagaaagca ctttcaaaac ttcacgagta tttccctgaa 2040
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tggatagagg gaagaggaaa atctgttgtt tgtgaagctg tcattccagc caaggttgtc 2160
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attactttaa tggaagcaag tggtcccaca aatgaagatt tatatatcag ctgcaccatg 2400
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cttatggcag cattggcagc aggacatctt gtcaaaagtc acatgattca caacaggtcg 180
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Claims (8)
1. A method for detecting serum anti-HMGCR antibody of immune necrotic myopathy, which is characterized by comprising the following steps:
s1: obtaining an HMGCR gene;
s2: constructing a pSJMY-mCherry recombinant fluorescent label vector;
s3: combining the HMGCR gene obtained in the step S1 with the recombinant fluorescent label vector obtained in the step S2 to construct a recombinant expression vector pSJMY-H-mCherry;
s4: transfecting the recombinant expression vector pSJMY-H-mCherry obtained in the step S3 with cells, and then carrying out immunoreaction on the HMGCR protein antigen expressed in the transfected cells and the serum to be detected for detecting whether an anti-HMGCR antibody exists in the serum to be detected.
2. The method of detecting serum anti-HMGCR antibodies against immune necrotizing myopathy of claim 1, wherein said S1 comprises the steps of:
1) extracting total RNA from patient tissues, and carrying out reverse transcription by taking the total RNA as a template to obtain total cDNA;
2) obtaining an HMGCR sequence from NCBI and dividing the HMGCR sequence into a front segment and a rear segment, designing a corresponding forward primer and a reverse primer, and carrying out PCR amplification on the cDNA obtained in the step 1) to obtain a corresponding HMGCR gene;
3) respectively carrying out TA cloning on the front fragment and the rear fragment of the HMGCR gene obtained in the step 2) and a T vector, and transforming the ligation product into a competent cell for transformation to obtain a transformant;
4) the transformant of step 3) is picked up for culture.
3. The method of claim 2, wherein the forward primer of the pre-segment of the HMGCR sequence of step 2) is GGGGCGATCGCCATGTTGTCAAGACTTTTTCGA, and the reverse primer of the pre-segment is TTTGCTGAGGTAGTAGGTTGGTCCACCACCCACCGTTCCTATCTCTATAGATGGCAT; the forward primer of the rear fragment of the HMGCR sequence was TTTCCTCAGCAAGCCTGTTTGCAGA, and the reverse primer of the rear fragment was AAAACGCGTGGCTGTCTTCTTGGTG.
4. The method of detecting serum anti-HMGCR antibodies against immune necrotizing myopathy of claim 1, wherein said S2 comprises the steps of:
5) carrying out PCR amplification on the mCherry red coral protein gene by using a primer pair;
6) constructing a pSJMY-mCherry recombinant fluorescent label vector by using the mChery red coral protein gene obtained in the step 5) and the vector pS 100010.
5. The method for detecting serum anti-HMGCR antibody against immune necrotizing myopathy as claimed in claim 4, wherein the mCherry red coral protein gene in step 5) has the forward primer of TACGCGGCCGCTCGAGATGGTGAGCAAGGGCGAGG and the reverse primer of CGCGGCCGGCCGTTTAAACCTACTTGTACAGCTCGTCCATGCC.
6. The method of detecting serum anti-HMGCR antibodies against immune necrotizing myopathy of claim 1, wherein said S3 comprises the steps of:
7) selecting transformants with correct sequencing of the front fragment and the rear fragment obtained in the step 4) for recombination, respectively cutting and recovering the front fragment and the rear fragment from a T carrier, connecting the front fragment and the rear fragment with the empty carrier pSJMY-mCherry containing mChery red coral protein fluorescence at the C end obtained in the step 6) to obtain pSJMY-H-mChery, and converting the connection product into escherichia coli to obtain the transformants;
8) performing colony PCR amplification on the transformant obtained in the step 7) by using a forward primer of the HMGCR front fragment and a reverse primer of the HMGCR rear fragment to determine a positive colony;
9) carrying out amplification culture and enzyme digestion identification on the positive colonies obtained in the step 8).
7. The method of detecting serum anti-HMGCR antibodies against immune necrotizing myopathy of claim 1, wherein said S4 comprises the steps of:
10) inoculating HEK293T cells into a culture system, transferring the pSJMY-H-mCherry vector obtained in the step 9) and a transfection reagent into the cells together when the concentration of the cells reaches 80%, and performing vector transfection to obtain the cells containing the recombinant expression vector pSJMY-H-mCherry;
11) further fixing, membrane breaking, coating and sealing the transfection vector cells obtained in the step 10);
12) adding a serum to be tested to the HMGCR protein antibody expressed by the cells obtained in the step 11) to perform immunoreaction detection.
8. The method for detecting serum anti-HMGCR antibody against necrotizing myopathy in accordance with claim 7, wherein said process of step 12) comprises:
12.1) diluting the serum sample in proportion;
12.2) discarding the confining liquid on the cell surface obtained in the step 11), adding the diluted serum obtained in the step 12.1) to obtain a uniformly mixed liquid, and incubating;
12.3) discarding the mixed solution in the step 12.2), adding a Triton-containing PBS washing solution into the mixed solution for washing, then adding a corresponding secondary antibody for incubation, and washing the secondary antibody with the Triton-containing PBS washing solution after the incubation is finished;
12.4) adding phosphate buffer solution to the substance obtained in the step 12.3) for immunofluorescence observation.
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