阅读说明：本技术 一种pET44a-scFv1956#表达载体的构建及其引物和应用 (Construction of pET44a-scFv1956# expression vector, primer and application thereof ) 是由 王长琛 潘博 李正勇 邢梦婷 蒋海越 魏斌 于 2020-04-29 设计创作，主要内容包括：本发明涉及抗体领域,具体涉及一种pET44a-scFv1956#表达载体的构建及其引物和应用。所述引物的序列为：引物1(44a up)：5’-TC CCC CGG GGC AGC CAG GTC CAA TTT GTA G-3’；引物2(44a down)：5’-CCG CTC GAG TTA ATT CAG ATC CTC TTC T-3’。本发明的pET44a-scFv1956#表达载体的构建方法能够顺利的构建表达载体。本发明pET44a-scFv1956#由抗体重链可变区(VH)-连接链(G4S1)3-轻链可变区(VL)构成,是没有Fc段的单价抗体,其大小是抗体全长的六分之一,能够解决现有的处理方式容易引起补体系统激活引起的神经肌肉接头免疫损伤的技术问题。由于pET44a-scFv1956#表达载体能够有利于抑制乙酰胆碱受体的抗体,所以可以用来制备抗皱纹药物,对皱纹有着良好的治疗效果。(The invention relates to the field of antibodies, in particular to construction of a pET44a-scFv1956# expression vector, and a primer and application thereof. The sequence of the primer is as follows: primer 1(44a up): 5'-TC CCC CGG GGC AGC CAG GTC CAA TTT GTA G-3', respectively; primer 2(44a down): 5'-CCG CTC GAG TTA ATT CAG ATC CTC TTC T-3' are provided. The method for constructing the pET44a-scFv1956# expression vector of the invention can successfully construct the expression vector. The pET44a-scFv1956# is composed of an antibody heavy chain variable region (VH) -connecting chain (G4S1) 3-light chain variable region (VL), is a monovalent antibody without Fc segment, has the size of one sixth of the whole length of the antibody, and can solve the technical problem that the existing treatment mode easily causes the neuromuscular junction immune injury caused by the activation of a complement system. Since the pET44a-scFv1956# expression vector can be beneficial to inhibiting the antibody of acetylcholine receptor, the vector can be used for preparing anti-wrinkle drugs and has good treatment effect on wrinkles.)

1. A primer for constructing a pET44a-scFv1956# expression vector is characterized in that the sequence of the primer is as follows:

primer 1(44a up): 5'-TC CCC CGG GGC AGC CAG GTC CAA TTT GTA G-3' the flow of the air in the air conditioner,

primer 2(44a down): 5'-CCG CTC GAG TTA ATT CAG ATC CTC TTC T-3', wherein:

the CCC CGG sequence is a restriction enzyme cutting site of Sma I;

the CTC GAG sequence is Xho I enzyme cutting site.

2. A method for constructing a pET44a-scFv1956# expression vector is characterized by comprising the following steps:

amplification of AChR scFv1956# structural Gene

1.1, using plasmid pHEN1 containing antihuman AChR scFv1956# as template, adding cleavage site Sma I at 5 'end and introducing cleavage site Xho I at 3' end by using primer 1 and primer 2 described in claim 1: carrying out PCR amplification on a required cloning fragment by Pyrobest high-fidelity DNA polymerase to obtain a product;

1.2, taking 5 mu L of the product obtained in the step 1.1 to perform 1% agarose gel electrophoresis analysis to obtain a required PCR product;

secondly, PCR recycling product and enzyme digestion of the fragment and the carrier

2.1, adding sodium acetate into the PCR product obtained in the step 1.2 to obtain a mixture, wherein the volume ratio of the PCR product to the sodium acetate is 10: 1;

2.2, adding ice-cold absolute ethyl alcohol with the volume 2 times that of the mixture in the step 2.1, uniformly mixing, placing on ice for 15-20min, and centrifuging to obtain a supernatant;

2.3, adding 70 wt% of ethanol into the supernatant obtained in the step 2.2, centrifuging, removing the supernatant, sucking the ethanol on the tube wall by using a filter paper strip, and airing at normal temperature to obtain a solid;

2.4, resuspending the solid with pure water to obtain a PCR recovery product, then sequentially carrying out enzyme digestion on the PCR recovery product and a pET44a (+) vector by SmaI (enzyme digestion temperature is 30 ℃) and Xho I, and recovering an enzyme digestion PCR fragment and a pET44a (+) vector after 8g/L agarose gel electrophoresis;

thirdly, the PCR fragment is connected with pET44a (+) carrier

3.1 adding the PCR fragment and pET44a (+) at 16 ℃ with T4 DNA ligase, ligating the PCR fragment with pET44a (+) vector to obtain a ligation product,

3.2, transforming 10 mu L of the ligation product into 100 mu L of competent JM109, standing for 24h, selecting positive clones, coating 100mg/L ampicillin 2 XYT agar plate, culturing for 24h, and extracting plasmid DNA;

3.3 DNA sequencing of the plasmid DNA obtained in step 3.2, the plasmid DNA was selected and sequenced correctly and named pET44a-scFv1956#, which contains a gene encoding a fusion protein of a single-chain antibody specifically binding to an acetylcholine receptor and complement regulatory factor (DAF),

construction of expression vector

4.1, digesting the pET44a-scFv1956# correctly sequenced in the step 3.3 by SmaI enzyme and Xho I enzyme, carrying out gel recovery on 1600bp DNA fragment, then transforming competent DH5 alpha cell, and then adding 2 XYT culture solution without antibody for 8h to obtain solution;

4.2, the solution obtained in step 4.1 was spread evenly on AMP + 2 × YT plates, IPTG was added at 37 ℃ until the logarithmic growth phase OD600 became 0.6, and after continuous induction for 4 hours, centrifugation was carried out, and the supernatant was discarded, thereby completing the construction of pET44a-scFv1956# expression vector.

3. The method for constructing pET44a-scFv1956# expression vector according to claim 2, wherein in step 2.2, the frequency of centrifugation is 2000-3000 r/min, and the time of centrifugation is 3-5 min.

4. The method for constructing pET44a-scFv1956# expression vector according to claim 2, wherein in step 2.3, the frequency of centrifugation is 1500-2000 r/min, and the time of centrifugation is 10-15 min.

5. The method for constructing pET44a-scFv1956# expression vector according to claim 2, wherein in step 4.2, the frequency of centrifugation is 1000-2000 r/min, and the time of centrifugation is 5-10 min.

6. An application of pET44a-scFv1956# expression vector in preparing anti-wrinkle medicine.

7. The application of pET44a-scFv1956# expression vector in preparing acetylcholine receptor antibody preparation.

Technical Field

The invention belongs to the field of antibodies, and particularly relates to construction of a pET44a-scFv1956# expression vector, and a primer and application thereof.

Background

The wrinkles are divided into postural wrinkles, dynamic wrinkles and gravitational wrinkles, wherein the dynamic wrinkles are the result of long-term contraction of the expression muscles and are mainly manifested in the eyebrow lifting lines of the frontal muscles, the glabellar lines of the frown muscles, the crow's feet lines of the orbicularis oculi muscles, the oral horn lines and the lip erections of the orbicularis oris muscles, the buccal twills of the zygomatic muscles and the upper labial quadratus, and the like. Effectively block the excessive contraction of the expression muscles, lead the expression muscles to relax and improve wrinkles. The signal transmission between the neuromuscular is dependent on calcium ion (Ca)²⁺) The specific area of the presynaptic membrane depolarizes when a nerve impulse is conducted to the motor nerve terminal, Ca²⁺Ca by voltage gating²⁺Channel influx, changes presynaptic membrane potential, attracts Ach (acetylcholine) -containing vesicles to the presynaptic membrane, and is released in a quantized fashion to the synaptic cleft. Ligand-gated Na of ACh and postsynaptic membrane⁺Transient increase in sodium ion (Na) upon binding of the acetylcholine receptor AChR (acetylcholine receptor) of the channel⁺) Permeability to give MEPP potential; when the motor nerve is stimulated, nerve impulse is transmitted to nerve endings to release a large amount of ACh molecules and more Na⁺Inflow when Na is abundant⁺Na with voltage-gated depolarization amplitude induced by influx⁺At the threshold of the channel, a positive feedback loop is formed, and the postsynaptic membrane depolarizes to generate AP, which causes muscle contraction. This process is very short and ACh is rapidIs degraded and inactivated by acetylcholinesterase. Normally, the more ACh is released by nerve impulses, the more AChR is activated.

AChR (adult type) at skeletal muscle NMJ is composed of 2^α5-mer consisting of β, -subunits α -subunit plays a key role in NMJ transmission, acetylcholine (ACh) and ACh R released by presynaptic membrane at NMJ^αOne of the 1-subunits currently has no very precisely located binding site (approximately amino acids 185 and 199 at the nitrogen terminus) that opens an ion channel, Na, in its center⁺The internal flow forms MEPP. The vast majority of ACh RAbs in patients with muscle weakness can interact with ACh R at NMJ^αA region of 64-76 subunits, the so-called Main Immune Region (MIR), is combined to cause transmission failure at NMJ. The antibody specifically binding to AChR is designed, can block the signal transmission of neuromuscular, can cause muscle contraction disorder locally, and can improve the wrinkle condition.

However, in the prior art, antibodies against acetylcholine receptors, which are pathologically pathogenic factors, in patients, such as T cells, immune cells, inflammatory cytokines and B cells, are often studied, and due to the cause of muscle contraction disorder, antibodies against acetylcholine receptors, which are pathological pathogenic factors in myasthenia gravis patients and animal models of myasthenia gravis, bind to acetylcholine receptors at neuromuscular junctions, activate a large amount of complement and deposit at the neuromuscular junctions to dissolve, and destroy postsynaptic membranes and acetylcholine receptors, resulting in transmission disorder at the neuromuscular junctions. Therefore, the existing treatment mode is easy to cause the immune injury of the neuromuscular junction caused by the activation of the complement system.

Disclosure of Invention

The invention provides a construction method of a pET44a-scFv1956# expression vector, a primer and an application thereof, aiming at solving the technical problem that the existing treatment mode in the background art easily causes neuromuscular junction immune damage caused by activation of a complement system.

The technical scheme adopted by the invention is as follows:

the invention provides a primer for constructing a pET44a-scFv1956# expression vector, which is characterized in that the sequence of the primer is as follows:

primer 1(44a up): 5'-TC CCC CGG GGC AGC CAG GTC CAA TTT GTA G-3' the flow of the air in the air conditioner,

primer 2(44a down): 5'-CCG CTC GAG TTA ATT CAG ATC CTC TTC T-3', wherein:

the CCC CGG sequence is a restriction enzyme cutting site of Sma I;

the CTC GAG sequence is Xho I enzyme cutting site.

The invention has the beneficial effects that: compared with the traditional antibody, the pET44a-scFv1956# of the invention is (1) structurally composed of an antibody heavy chain variable region (VH) -connecting chain (G4S1) 3-light chain variable region (VL), is a monovalent antibody without Fc segment, and has the size of one sixth of the whole length of the antibody; (2) the acetylcholine receptor single-chain antibody only has 1 antigen binding valency, can only be combined with one main immunogen region in two alpha-subunits on the acetylcholine receptor, and cannot cause antigen modulation caused by crosslinking between acetylcholine receptor molecules; (3) in addition, the acetylcholine receptor single-chain antibody has no Fc segment, so that complement can not be activated, and immune damage caused by activation of a complement system is avoided; (4) the acetylcholine receptor single-chain antibody can be specifically combined with an acetylcholine receptor, and can block and block the combination of an acetylcholine receptor antibody, namely a pathogenic factor in a patient with myasthenia gravis, and the acetylcholine receptor antibody is combined with the acetylcholine receptor, so that the acetylcholine receptor is protected from being attacked by the pathogenic acetylcholine receptor antibody in the patient with myasthenia gravis, and the single-chain antibody is an ideal targeting tool due to the characteristic. Therefore, the pET44a-scFv1956# expression vector of the present invention makes it possible to obtain purified soluble anti-human acetylcholine receptor single-chain antibody 1956 #. Can solve the technical problem that the existing treatment mode easily causes the immune injury of the neuromuscular junction caused by the activation of a complement system. The primers for construction of the pET44a-scFv1956# expression vector are necessary for construction of the pET44a-scFv1956# expression vector.

In another aspect, the present invention provides a method for constructing pET44a-scFv1956# expression vector, comprising the following steps:

amplification of AChR scFv1956# structural Gene

1.1, using plasmid pHEN1 containing antihuman AChR scFv1956# as template, adding cleavage site Sma I to 5 'end of primer 1 and primer 2, and introducing cleavage site Xho I to 3' end: carrying out PCR amplification on a required cloning fragment by Pyrobest high-fidelity DNA polymerase to obtain a product;

1.2, taking 5 mu L of the product obtained in the step 1.1 to perform 1% agarose gel electrophoresis analysis to obtain a required PCR product;

secondly, PCR recycling product and enzyme digestion of the fragment and the carrier

2.1, adding sodium acetate into the PCR product obtained in the step 1.2 to obtain a mixture, wherein the volume ratio of the PCR product to the sodium acetate is 10: 1;

2.2, adding ice-cold absolute ethyl alcohol with the volume 2 times that of the mixture in the step 2.1, uniformly mixing, placing on ice for 15-20min, and centrifuging to obtain a supernatant;

2.3, adding 70 wt% of ethanol into the supernatant obtained in the step 2.2, centrifuging, removing the supernatant, sucking the ethanol on the tube wall by using a filter paper strip, and airing at normal temperature to obtain a solid;

2.4, resuspending the solid with pure water to obtain a PCR recovery product, then sequentially carrying out enzyme digestion on the PCR recovery product and a pET44a (+) vector by using Sma I (enzyme digestion temperature is 30 ℃) and Xho I, and carrying out agarose gel electrophoresis at 8g/L to recover an enzyme digested PCR fragment and a pET44a (+) vector;

thirdly, the PCR fragment is connected with pET44a (+) carrier

3.1 adding the PCR fragment and pET44a (+) with T4 DNA ligase at 16 ℃ and ligating the PCR fragment with pET44a (+) vector to obtain a ligation product

3.2, transforming 10 mu L of the ligation product into 100 mu L of competent JM109, standing for 24h, selecting positive clones, coating 100mg/L ampicillin 2 XYT agar plate, culturing for 24h, and extracting plasmid DNA;

3.3, the plasmid DNA obtained in step 3.2 was subjected to DNA sequencing analysis, and the DNA was selected and sequenced correctly and named pET44a-scFv1956#, which contains a gene encoding a fusion protein of a single-chain antibody specifically binding to an acetylcholine receptor and complement regulatory factor (DAF).

Construction of expression vector

4.1, digesting the pET44a-scFv1956# correctly sequenced in the step 3.3 by SmaI enzyme and Xho I enzyme, carrying out gel recovery on 1600bp DNA fragment, then transforming competent DH5 alpha cell, and then adding 2 XYT culture solution without antibody for 8h to obtain solution;

4.2, the solution obtained in step 4.1 was spread evenly on AMP + 2 × YT plates, I PTG was added at 37 ℃ until the logarithmic growth phase OD600 became 0.6, the induction was continued for 4 hours, and the supernatant was centrifuged to discard the supernatant, thereby completing the construction of pET44a-scFv1956# expression vector.

The invention has the advantages that the pET44a-scFv1956# expression vector can be successfully obtained by the construction method of the pET44a-scFv1956# expression vector, thereby clearing obstacles for the subsequent obtaining of the purified soluble anti-human acetylcholine receptor single-chain antibody 1956 #. Can solve the technical problem that the existing treatment mode easily causes the immune injury of the neuromuscular junction caused by the activation of a complement system.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, in step 2.2, the centrifugation frequency is 2000-3000 r/min, and the centrifugation time is 3-5 min.

The beneficial effect of adopting the further scheme is that the determined parameters are beneficial to the completeness of centrifugation.

Further, in step 2.3, the centrifugation frequency is 1500-2000 r/min, and the centrifugation time is 10-15 min.

The further scheme has the beneficial effects that the determined parameters are beneficial to thorough centrifugation, and purer separated substances are obtained.

Further, in the step 4.2, the centrifugation frequency is 1000-2000 r/min, and the centrifugation time is 5-10 min.

The further scheme has the beneficial effects that the determined parameters are beneficial to thorough centrifugation, and purer separated substances are obtained.

The invention also provides application of the pET44a-scFv1956# expression vector in preparing an anti-wrinkle medicament.

The invention has the beneficial effect that the pET44a-scFv1956# expression vector can be beneficial to inhibiting the antibody of acetylcholine receptor, so that the vector can be used for preparing anti-wrinkle drugs. And the wrinkle-resisting powder can obtain a good wrinkle-treating effect, fills the gap of lacking anti-wrinkle medicines in the market, greatly promotes the improvement of the living standard of people, and meets the beauty-loving requirements of people.

The invention also provides application of the pET44a-scFv1956# expression vector in preparation of an antibody preparation of an acetylcholine receptor.

The pET44a-scFv1956# expression vector can produce acetylcholine receptor antibody, prevent symptoms such as myasthenia and the like, and prevent wrinkles.

Drawings

FIG. 1 is a diagram showing the result of agarose gel electrophoresis detection of the PCR product of the present invention, in which the sequence number 1 is the PCR product and the sequence number 2 is DNAmarker.

FIG. 2 is a diagram showing the results of agarose gel electrophoresis detection of the products of PCR recovery and pET44a (+) vector of the present invention digested with SmaI (digestion temperature 30 ℃ C.), Xho I.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.