CN116004639A - An N-terminal fragment of Glypican 3, an encoding gene, a polyclonal antibody, its preparation method and application - Google Patents

Abstract

The application relates to the field of immunoassay, in particular to an N-terminal fragment of glypican 3, a coding gene, a polyclonal antibody, a preparation method and application thereof; the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1; the amino acid sequence of the N-terminal fragment protein is shown as SEQ ID NO. 2; the polyclonal antibody specifically binds to the N-terminal fragment protein; by designing the nucleotide sequence of the coding gene of the N-terminal fragment protein of the glypican 3 and a protein preparation method, the method is similar to the natural GPC3 in the modification folding stage, the folding modification accuracy of the N-terminal fragment protein is improved, and the mRNA formed in the transcription stage has no complex secondary structure due to the simpler 5' -end of the designed nucleotide sequence, so that the difficulty in the translation stage is reduced, the translation efficiency is improved, and the expression quantity of the N-terminal fragment protein is improved.

Description

N-terminal fragment of Glypican 3, coding gene, polyclonal antibody, preparation method and application thereof

technical field

The present application relates to the field of immune analysis, in particular to an N-terminal fragment of Glypican 3, a coding gene, a polyclonal antibody, and a preparation method and application thereof.

Background technique

Glypican 3 (GPC3) is a family of heparan sulfate glycoproteins, involved in the regulation of individual development, cell proliferation and differentiation, among them, Glypican 3 (Glypican-3, GPC3 ) was first cloned from a human embryonic cDNA library when Pillia et al. studied Simpson-Golabi-Behmel syndrome (SGBS) in 1996. The gene encoding GPC3 is located on human chromosome Xq26[2-3], and its structure is more than 900kb in length. It is one of the largest human genes; the 5' end of the gene faces the telomeric region, and the 3' end faces the centriole region. It consists of 8 exons and 7 introns, and there are many transcription factor binding sites in the promoter region of the gene. At the same time, the full length of the cDNA sequence of GPC3 is 2263bp, including an open reading frame of 1740bp and 580 amino acids encoded by the open reading frame, and the molecular weight of the expressed product is about 66KDa.

Since GPC3 is closely related to the occurrence and development of various tumors, especially highly expressed in hepatocellular carcinoma, it is a potential important serological and histological diagnostic marker for liver cancer, so the research on GPC3 is very necessary. According to the existing research, although the GPC3 protein content in the blood is not significantly correlated with the mRNA expression in the tissue, the increase and change of the protein content in the blood still reflect that the primary tumor of the liver is hepatocellular carcinoma (HCC). Therefore, the development of GPC3 protein detection technology is very necessary for the development of liver cancer diagnosis, but it is very difficult to detect serum secreted GPC3 with antibodies that recognize the C-terminal (359-554AA) fragment of GPC3. Most of the stages used antibodies to the N-terminal 25-358 region of GPC3 to detect serum secreted forms of GPC3.

At present, the N-terminal protein of GPC3 is traditionally expressed and prepared using 25-358AA of NP_001158089.1, which comes from glypican-3isoform 1, and is expressed in a prokaryotic or eukaryotic system. However, although the expression level in the eukaryotic system is high, but The modified folding structure is incorrect, which affects the accuracy of detection, and the expression level in the prokaryotic system expression process is not high, so it is difficult to be clearly detected; therefore, how to increase the expression level of the N-terminal protein of GPC3 while improving the accuracy of folding modification It is a technical problem that needs to be solved urgently.

Contents of the invention

This application provides an N-terminal fragment of Glypican 3, a coding gene, a polyclonal antibody and its preparation method and application, so as to solve the problem of the expression level and folding modification of the N-terminal protein of GPC3 in the prior art The technical problem that the accuracy is difficult to improve at the same time.

In a first aspect, the application provides a gene encoding an N-terminal fragment, the nucleotide sequence of the gene encoding is shown in SEQ ID NO:1.

In the second aspect, the present application provides an N-terminal fragment protein of Glypican 3, the N-terminal fragment protein is transcribed and translated from the coding gene described in the first aspect.

Optionally, the amino acid sequence of the N-terminal fragment protein is shown in SEQ ID NO:2.

In a third aspect, the present application provides a recombinant vector, which includes the coding gene described in the first aspect.

Optionally, the recombinant vector includes a eukaryotic vector.

In a fourth aspect, the present application provides a mammalian expression cell, which includes the recombinant vector described in the third aspect.

In the fifth aspect, the present application provides a method for preparing an N-terminal fragment protein, the preparation method:

Synthesizing the coding gene described in the first aspect;

Constructing the recombinant vector described in the third aspect with the coding gene;

transfecting competent cells with the recombinant vector, and then culturing to obtain the expression product of the N-terminal fragment;

The expression product is separated and then purified to obtain the N-terminal fragment protein.

In a sixth aspect, the present application provides a polyclonal antibody of an N-terminal fragment protein, and the polyclonal antibody specifically binds to the N-terminal fragment protein described in the second aspect.

In the seventh aspect, the present application provides a method for preparing the polyclonal antibody described in the sixth aspect, the method comprising:

Immunizing the animal model with the N-terminal fragment protein described in the second aspect to obtain primary polyclonal antibodies;

The primary polyclonal antibody is subjected to antigen affinity purification to obtain specific polyclonal antibody.

In the eighth aspect, the present application provides an application of the N-terminal fragment of Glypican 3, characterized in that the application includes:

The N-terminal fragment protein described in the second aspect is used in the preparation of the kit.

Compared with the prior art, the above-mentioned technical solutions provided by the embodiments of the present application have the following advantages:

The coding gene of a kind of N-terminal fragment protein provided in the embodiment of the present application, by designing the nucleotide sequence of the gene encoding the N-terminal fragment protein of Glypican 3, due to the 5' of the designed nucleotide sequence The end is relatively simple, so that the mRNA formed in the transcription stage has no complex secondary structure, thereby reducing the difficulty of the translation stage, improving translation efficiency, and increasing the expression of the N-terminal fragment protein. Since the eukaryotic mammalian cell expression system is used, it can effectively carry out glycosylation modification and disulfide bond formation, so it is similar to the natural GPC3 in the modification and folding stage, which can improve the folding modification efficiency of the N-terminal fragment protein accuracy.

Description of drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description serve to explain the principles of the invention.

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, In other words, other drawings can also be obtained from these drawings without paying creative labor.

Figure 1 is a schematic flow diagram of the preparation method of the N-terminal fragment protein provided in the examples of the present application;

Figure 2 is a schematic flow diagram of the preparation method of the polyclonal antibody provided in the embodiment of the present application;

Figure 3 is the agarose gel electrophoresis image of the double-digestion product provided in the embodiment of the present application, wherein, 1 is the double-digestion product with V5H vector cloning enzyme cleavage site added at both ends of NM_004484218-1807bp, and 2 is the double-digestion product of NM_004484 218-1219bp 3 is the double digestion product of NM_004484 218-1219bp with PET30a+ vector cloning restriction site added at both ends, 4 is the double digestion product of NM_001164619.2 218-1057bp with V5H vector added at both ends Cloning of the double-digestion product of the restriction site;

Fig. 4 is the sequence diagram of the recombinant plasmid inserted into NM_004484 218-1807bp in the V5H recombinant vector provided by the embodiment of the present application;

Fig. 5 is the sequence diagram of the recombinant plasmid inserted into NM_004484 218-1219bp in the V5H recombinant vector provided by the embodiment of the present application;

Fig. 6 is the sequence map of the recombinant plasmid inserted into NM_004484 218-1219bp in the PET30a+ recombinant vector provided by the embodiment of the present application;

Fig. 7 is the sequence map of the recombinant plasmid inserted into NM_001164619.2 218-1057bp in the V5H recombinant vector provided by the embodiment of the present application;

Fig. 8 is the SDS-PAGE electrophoresis figure of the eukaryotic expression GPC3 protein fragment provided by the embodiment of the present application, wherein, 4-6 is NP_001158089.1 (glypican-3isoform 1) 25-358AA fragment, and 7-9 is NP_001158091.1 ( glypican-3isoform 4) 25-304AA fragment, 1, 4 and 7 are the whole supernatant of eukaryotic expression cells, 2, 5 and 8 are the effluent after loading the whole supernatant, 3, 6 and 9 are Eluted protein;

Fig. 9 is the SDS-PAGE electrophoresis figure of the prokaryotic expression GPC3 protein fragment provided by the embodiment of the present application, wherein, the protein fragment is (NP_001158089.1, glypican-3isoform 1, 25-358AA fragment);

Figure 10 is a diagram of the detection results of eukaryotic cells expressing the GPC3 protein fragment cell supernatant WB provided by the embodiment of the present application, wherein, 1 is the detection result of NP_001158089.1 (glypican-3isoform 1) 25-559AA fragment, and 2 is NP_001158089. 1 (glypican-3isoform 1) 25-358AA fragment detection result, 3 is the detection result of NP_001158091.1 (glypican-3isoform 4) 25-304AA fragment.

Fig. 11 is a diagram of the results of polyclonal antibody immunohistochemical detection of normal liver tissue provided in the examples of the present application.

Fig. 12 is a graph showing the results of multi-antibody immunohistochemical detection of HCC cancer tissues provided in the examples of the present application.

Figure 13 is a standard curve diagram provided by the present application example.

Detailed ways

The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be presented more clearly. Those skilled in the art should understand that these specific implementations and examples are used to illustrate the present invention, not to limit the present invention.

Throughout the specification, unless otherwise specified, terms used herein should be understood as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, this specification shall take precedence.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

The creative thinking of this application is:

Since the secretion of GPC3 in the blood may have several forms: 1. The site between the arginine at position 358 and the serine at position 359 is cut by Furin (a protease) to form an N-terminal protein 40KDa.

II. A glycosylated form with a molecular weight greater than 100 kDa through notum-mediated cleavage of the GPI anchor site;

III. A 50 kDa fragment lacking the HS side chain;

IV. Since soluble GPC3 can be detected as a shorter form in human HCC cell culture supernatants, there may also be alternative shedding or lyases that require further analysis.

Therefore, it was not until after 2003 that the research on GPC3 antibodies began to mature and GPC-3 immunoassay technology appeared, including ELISA quantitative detection technology.

Although the GPC3 protein content in the blood is not significantly correlated with the mRNA expression in the tissue, the increase and change of the protein content in the blood can still reflect the occurrence and development of the primary tumor of the liver, which is hepatocellular carcinoma (HCC). The main rationale for this may be that the N-terminal form of the GPC3 protein enters the blood circulation, but it appears to be very difficult to detect serum secreted forms of GPC3 using antibodies that recognize the C-terminal (359-554AA) fragment of GPC3. Antibodies in the 25-358 interval are relatively easy to enter.

At present, the N-terminal protein of GPC3 is traditionally expressed and prepared using 25-358AA of NP_001158089.1, which comes from glypican-3isoform 1, and is expressed in a prokaryotic or eukaryotic system. However, although the expression level in the prokaryotic system is high, the modification The folding structure is incorrect, which affects the accuracy of detection. This is mainly because when designing the gene encoding the N-terminal protein, the codon is optimized for the sake of expression, but the specific site is deleted at the same time, so that The modified folding structure is incorrect; the expression level in the eukaryotic system expression process is not high, and it is difficult to be clearly detected, mainly because the gene encoding the N-terminal protein is only considered in the prokaryotic system when designing and optimizing Codon coding optimization does not take into account the complex secondary structure of mRNA in the translation phase, resulting in insufficient expression of the N-segment protein in the translation phase.

Therefore, how to increase the expression level of the N-terminal protein of GPC3 and at the same time improve the accuracy of folding modification is a technical problem that needs to be solved urgently.

The technical solutions provided by the embodiments of the present invention are to solve the above-mentioned technical problems, and the general idea is as follows:

In one embodiment of the present application, a gene encoding an N-terminal fragment is provided, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:1.

Next, an N-terminal fragment protein of Glypican 3 provided in the embodiment of the present application is described, and the N-terminal fragment protein is transcribed and translated from the coding gene.

Since the coding gene encoding the amino acid sequence of the N-terminal fragment protein described in the embodiment of the present application is the coding gene provided above in the embodiment of the present application, the nucleotide sequence and composition information of the coding gene will not be repeated here. All genes encoding the N-terminal fragment proteins of the embodiments of the present application belong to the scope of protection of the present application.

In some optional embodiments, the amino acid sequence of the N-terminal fragment protein is shown in SEQ ID NO:2.

In the examples of the present application, the specific composition of the amino acid sequence of the N-terminal fragment protein is controlled, and the designed amino acid sequence can effectively improve the expression level of the N-terminal fragment protein and the accuracy of modified folding.

Next, a recombinant vector provided in the embodiment of the present application is described, the recombinant vector includes the coding gene.

Since the recombinant vector introduced in the embodiment of the present application includes the coding gene provided above in the embodiment of the present application, the nucleotide sequence and composition information of the coding gene will not be repeated here. All recombinant vectors including the coding genes of the embodiments of the present application belong to the scope of protection of the present application.

In some optional embodiments, the recombinant vector includes a eukaryotic vector, wherein the eukaryotic vector may be a V5H vector.

In the embodiment of the present application, the type of the recombinant vector is controlled so that it can fully transfer the coding gene to competent cells in the eukaryotic expression system, paving the way for subsequent transcription and translation.

Next, a mammalian expression cell provided in the embodiment of the present application is described, and the expression cell includes the recombinant vector.

Since the expression cells described in the examples of the present application contain the recombinant vectors provided above in the examples of the present application, the composition information of the recombinant vectors will not be repeated here. All expression cells including the recombinant vectors of the embodiments of the present application belong to the scope of protection of the present application.

Next, a method for preparing an N-terminal fragment protein provided in an embodiment of the present application is described, the preparation method:

S1. Synthesizing the coding gene;

S2. Constructing the recombinant vector with the coding gene;

S3 transfecting competent cells with the recombinant vector, and then culturing to obtain the expression product of the N-terminal fragment;

S4. Separating and purifying the expression product to obtain the N-terminal fragment protein.

Since the preparation method described in the examples of the present application targets the N-terminal fragment protein as provided above in the examples of the present application, the amino acid sequence information of the N-terminal fragment protein and the nucleotides encoding the gene will not be repeated here. acid sequence. All the preparation methods of the N-terminal fragment proteins including the examples of the present application belong to the scope of protection of the present application.

Next, a polyclonal antibody to an N-terminal fragment protein provided in an embodiment of the present application is described, and the polyclonal antibody specifically binds to the N-terminal fragment protein.

Since the N-terminal fragment protein of the polyclonal antibody described in the embodiment of the present application is the N-terminal fragment protein provided in the foregoing embodiment of the present application, the composition information of the N-terminal fragment protein will not be repeated here. All polyclonal antibodies comprising the N-terminal fragment proteins of the embodiments of the present application belong to the scope of protection of the present application.

Next, a method for preparing a polyclonal antibody to an N-terminal fragment protein provided in an embodiment of the present application is described, and the method includes:

S1. Using the N-terminal fragment protein as an immunogen, according to a conventional procedure, immunize an animal model;

S2. Measure the titer and collect the serum of the immune animal;

S3. Perform antigen affinity purification on the primary polyclonal antibody to obtain specific polyclonal antibody.

Since the preparation method described in the examples of the present application targets the polyclonal antibodies provided above in the examples of the present application, the composition information of the polyclonal antibodies will not be repeated here. All preparation methods of polyclonal antibodies including the examples of this application belong to the scope of protection of this application.

Next, the application of an N-terminal fragment of Glypican 3 provided in the embodiment of the present application is described, and the application includes:

The N-terminal fragment protein is used in the preparation of the kit.

Due to the application described in the embodiment of the present application, the N-terminal fragment is the N-terminal fragment protein provided above in the embodiment of the present application, so the amino acid sequence information of the N-terminal fragment protein will not be repeated here. All applications including the N-terminal fragment proteins of the embodiments of the present application belong to the scope of protection of the present application.

Example 1

Synthesis of genes and construction of recombinant expression plasmids:

Gene synthesis:

1. Synthesize NM_001164619.2 218-1057bp (corresponding to NP_001158091.1, glypican-3isoform 4, 25-304AA), and add V5H vector cloning enzyme cutting site NheI/BamHI at both ends of the synthesized gene;

2. Synthesize NM_004484 218-1219bp (NP_001158089.1, glypican-3isoform 1, 25-358AA), and add V5H vector cloning enzyme cutting site NheI/BamHI at both ends of the synthesized gene;

3. Synthesize NM_004484 218-1219bp (NP_001158089.1, glypican-3isoform 1, 25-358AA), and add PET30a+ vector cloning enzyme cutting site BamHI/XhoI at both ends of the synthesized gene;

4. Synthesize NM_004484 218-1807bp (NP_001158089.1, glypican-3isoform 1, 25-554AA), and add V5H vector cloning restriction site NheI/BamHI at both ends of the synthesized gene.

5. Carry out NheI/BamHI double digestion of the synthesized gene sequence with the endonuclease of the added restriction site, the vector V5H is double-digested by the endonuclease NheI/BamHI, the vector PET30a+ is subjected to the endonuclease BamHI/XhoI Double enzyme digestion, the product was gel-cut and recovered after agarose gel electrophoresis, and the double enzyme digestion product was obtained, the results are shown in Figure 3.

6. The double-digested product was ligated overnight in a 16°C water bath with T4 ligase, transformed into E.coli DH5α strain, and positive clones were screened.

After sequencing and identification, as shown in Figure 2, the results showed that the inserted fragment was completely consistent with the fragment in the published full sequence, and inserted into the cloning site of the expression vector in the correct direction, as shown in Figure 4, Figure 5, and 6 and Figure 7. Four recombinant expression plasmids V5H-25-304AA, V5H-25-358AA, V5H-25-559AA and PET30a+-25-358AA were amplified and extracted.

Example 2

Embodiment 2 is compared with embodiment 1, and the difference between embodiment 2 and embodiment 1 is:

Expression and purification of eukaryotic fusion proteins:

1. Culture HEK293 cells in good condition in a 6-well plate at a density of 2×105 cells/well. After 24 hours of culture, transfect when the cell confluence is 80% to 90%. Cells are transfected with PBS Wash 3 times, add serum-free DMEM high-glucose medium, carry out according to the instructions of LipofectamineTM2000 transfection reagent, and transfect the three obtained recombinant expression plasmids V5H-25-304AA, V5H-25-358AA and V5H-25-554AA respectively , cultured at 37° C. and 5% CO 2 for 72 hours at a speed of 120 rpm, then collected the cell culture solution, centrifuged at 4500 g for 15 minutes, removed the cells, and took the supernatant.

2. Take 1mL of Ni-NTA Agarose affinity filler and pack it into the column, equilibrate the Ni-NTA affinity column for 10 column volumes with equilibration buffer (50mM PB, 0.3M NaCl, 10mM imidazole, pH 8.0), The supernatant of the centrifuged cell culture solution was passed through the Ni-NTA affinity column at a flow rate of 1 mL/min, and the flow-through was collected and stored at 4°C.

3. Wash 10 column volumes with washing buffer 1 (50mM PB, 0.3M NaCl, 20mM imidazole, pH8.0), collect the flow-through and store at 4°C. Wash 4-5 column volumes with elution buffer (50mM PB, 0.3M NaCl, 250mM imidazole, pH8.0), collect the eluate, and wash in dialysate (50mM PB, pH7.8, 0.3M NaCl, 5% Glycerol) was dialyzed overnight at 4°C to obtain GPC3 protein, and a small amount of GPC3 protein was subjected to SDS PAGE electrophoresis, and the results are shown in Figure 8.

Example 3

Embodiment 3 is compared with embodiment 2, and the difference between embodiment 3 and embodiment 2 is:

Expression, purification and renaturation of prokaryotic fusion proteins:

1. Add 10 μL of PET30a+-25-358AA plasmid into 100 μL of competent BL21 bacteria, place on ice for 20 minutes; then heat shock at 42°C for 90 seconds, quickly place on ice for 5 minutes, add 600 μL of LB culture medium; Shake at 37° C. and 220 rpm for 1 h, and then centrifuge. All the centrifuged supernatants are spread on LB plates containing kana, and cultured upside down at 37° C. overnight.

2. Pick the single clone on the transformation plate and inoculate it in a test tube containing 3 mL of kana LB culture medium, shake it overnight at 37°C at a speed of 220 rpm; the next day, inoculate it at a volume ratio of 1:100 in a tube containing In the 30mL LB culture medium of kana, shake at 220rpm at 37℃ for about 2h until the OD600 of the bacteria is 0.6-0.8; take out 1mL of the culture, centrifuge at 10000g for 2min at room temperature, discard the supernatant, and use 100μL of The bacterial pellet was resuspended in 1× loading buffer; IPTG was added to the remaining cultures to a final concentration of 0.5 mM, and then induced by shaking at 11°C and 220 rpm for 12 hours to express the fusion protein.

3. Centrifuge the culture solution after induced expression at 6000g for 10min at low temperature, then add 20mL of RIPA lysate to resuspend the bacterial pellet, and ultrasonically disrupt (power 400W, work for 4s, intermittent for 8s, 20min in total); The cell lysate was centrifuged at 10000 g for 20 min at 4 °C to collect the precipitate.

4. Use the inclusion body washing solution (20mM Tris, 1mM EDTA, 2M urea, 1M NaCl, 1% Triton X-100, pH 8.0) to wash the inclusion body 3 times for the precipitate, and then dissolve it with Buffer solution (20mM Tris, 5mM DTT, 8M urea, pH 8.0) dissolves inclusion bodies, sonicate (power 400W, work for 4s, rest for 8s, total 20min), and the product of the second sonication is placed at 4°C Centrifuge at 10,000 g for 20 min to collect the supernatant.

5. Get 1mL of Ni-NTA Agarose affinity filler to pack into the column, equilibrate the Ni-NTA affinity column with equilibration buffer (20mM Tris, 5mM DTT, 8M urea, pH 8.0) for 10 column volumes, and put The ultrasonic supernatant of the inclusion body was passed through the Ni-NTA affinity column at a flow rate of 1 mL/min, and the flow-through was collected and stored at 4°C, and then washed with washing buffer (20 mM Tris, 5 mM DTT, 8 M urea pH 8.0) for 10 column volume, collect the flow-through solution and store it at 4°C, wash with elution buffer (20mM Tris, 5mM DTT, 8M urea, 250mM imidazole, pH 8.0) for 4-5 column volumes, collect and wash Remove liquid, dialyze in dialysate (50mM PB, pH 7.8, 0.3M NaCl, 5% glycerol) overnight at 4°C to obtain GPC3 protein, take a small amount for SDS PAGE electrophoresis, the result is shown in Figure 9 shown.

Example 4

Embodiment 4 is compared with embodiment 3, and the difference between embodiment 4 and embodiment 3 is:

ELISA indirect method for detection of recombinant protein:

1. Antigen coating: Carbonate buffer was used as the coating solution, and the original coating was GPC3 recombinant protein at a concentration of 1.5 μg/mL. Put 100 μL in each well of a 96-well microtiter plate, overnight at 4°C, and then wash. Return the coated plate to room temperature, pour off the coating solution, add 300 μL of washing solution to each well, shake for 1 min each time, wash 3 to 4 times, and pat dry;

2. Blocking: add 200 μL calf serum with a mass concentration of 10% to each well as a blocking solution, incubate at 37°C for 1 hour; wash, return to room temperature, pour off the blocking solution, wash three times, shake for 1 min each time, and pat dry;

3. Dilute the GPC3 antibody with a buffer solution to a concentration of 5 μg/mL, add 100 μL to each well, and set a blank control well (PBS), and place it at 37 ° C for 30 min; wash 3 times, shake for 1 min each time, and pat dry;

4. Add enzyme-labeled secondary antibody: add 100 μL of HRP enzyme-labeled goat anti-mouse IgG diluted in a mass ratio of 1:10000 to each well, place at 37°C for 30 minutes; wash 3 times, shake for 1 minute each time, and pat dry;

5. Color development: add 00 μL of substrate chromogenic solution 1 to each well, and incubate at 37°C in the dark for 15 minutes; to terminate the reaction, add 50 μL of stop solution to each well.

6. Terminate the reaction; measure the OD450nm value, and read the optical density value of each well with a microplate reader whose detection wavelength is 450nm.

Among them, the OD450nm value of the negative control well was set as N, the positive as P, and P/N≧2.1 as the positive result. The results are shown in Table 1.

Table 1

It can be seen from Table 1 that the results of eukaryotic fragments 25-304A, 25-358AA and 25-554AA are relatively consistent, among which eukaryotic 25-554AA is the full-length GPC3 with the highest detection rate, and prokaryotic 25-358AA is basically not positive, so it is possible There is the problem of modifying the conformation.

Example 5

Embodiment 5 is compared with embodiment 4, and the difference between embodiment 5 and embodiment 4 is:

Eukaryotic recombinant protein expression:

1. Collect 1 mL of the supernatant of HEK293 cells transfected with the three eukaryotic expression plasmids V5H--25-304AA, V5H--25-358AA and V5H--25-559AA.

2. Take 80 μL of the supernatant of the three types of cells, add 20 μL of 5X sample buffer, boil for 5 minutes in an induction cooker, and then add 4% to 12% of the mass concentration of the Bis-Tris gel plate into the sample well, The antigenic protein is separated by electrophoresis to obtain a gel plate containing the antigenic protein.

3. Use refrigerated transfer buffer to transfer the antigenic protein to the fibrous membrane, then place the membrane with the transferred antigenic protein in the blocking solution and incubate on a shaker, then take out the membrane and place it in the blocking solution at a mass ratio of 1 : 100-1:500 diluted GPC3 polyclonal antibody (purchased from Mitaka Biotech), and incubated with shaking. Transfer the membrane to horseradish peroxidase-labeled goat anti-horse secondary antibody conjugate diluted with blocking solution at a mass ratio of 1:5000 to 1:10000, and incubate with shaking.

4. Place the membrane in the substrate and keep it away from light to obtain a membrane containing the substrate incubator, then expose and develop. The results are shown in Figure 10.

It can be seen from Figure 10 that the eukaryotic fragment 25-554AA has obvious degradation, and there are 3 bands, 70, 40 and 30kDa, with a high expression level; the eukaryotic fragment 25-304A has a high expression level and no obvious degradation; Nuclear fragment 25-358AA was not significantly expressed.

Example 6

Embodiment 6 is compared with embodiment 5, and the difference between embodiment 6 and embodiment 5 is:

Preparation of polyclonal antibodies:

1. Using the N-terminal fragment recombinant protein of GPC3 (NP_001158091.1, glypican-3isoform 4, 25-304AA) as the immunization antigen, the specific amino acid sequence is shown in SEQ ID NO: 2, and immunized 2 New Zealand white rabbits (body weight 2kg ～2.5kg), immunized according to standard immunization procedures, including: the first immunization was mixed with equal volume of recombinant protein and complete Freund's adjuvant, subcutaneous immunization, the dose was 400 μg/time, and 3 weeks later, equal volume of recombinant protein and no Complete Freund's adjuvant was mixed, the second subcutaneous immunization, the dose of recombinant protein immunization was the same as the first immunization, after that, immunization once every two weeks, same as the second immunization, until a total of 5-6 immunizations.

2. Blood collection test, determine the titer of the antiserum against the recombinant protein of the N-terminal fragment of GPC3 by indirect ELISA method, wait for the titer to be greater than 1:100,000 to prepare antiserum, and obtain polyantibody by antigen affinity purification.

Example 7

Embodiment 7 is compared with embodiment 6, and the difference between embodiment 7 and embodiment 6 is:

Multiple antibody immunohistochemical detection:

1. Dewaxing and hydration: Put the dried paraffin slices into fresh xylene, soak for 10 minutes, replace the dye vat and repeat twice; the slices are hydrated with alcohol, including: soaking in absolute ethanol for 5 minutes, soaking in 95% ethanol for 2 minutes times (2 minutes each time), soak in 85% ethanol for 2 minutes; soak in 75% ethanol for 2 minutes, rinse with tap water, and wash with deionized water for 2 minutes × 2 times;

2. Eliminate endogenous catalase: incubate at room temperature with 2% to 3% hydrogen peroxide (diluted in 0.01M PBS) for 5min to 10min to eliminate the effect of endogenous catalase, then distilled water Wash the film 3 times;

3. Antigen retrieval: heat 0.01M citric acid buffer (pH 6.0) in a beaker to 100°C, put slices in and keep at 95°C-100°C for 2min-3min for heat restoration, wait for antigen restoration The solution was naturally cooled to room temperature or indirectly cooled to room temperature through a cold water bath, and the specimen was washed 3 times with 0.01M PBS, each time for 1-3 minutes.

4. Polyclonal antibody incubation: add one drop of diluted polyclonal antibody working solution (20 μL to 50 μL) to the slide tissue, and incubate at room temperature for 2 hours or at 37 ° C for 60 minutes.

5. Washing: wash the specimen with 0.01M PBS for 3 times, each time for 1-3 minutes.

6. Secondary antibody incubation: add one drop of secondary antibody working solution (20 μL to 50 μL) to the slide tissue, and incubate at room temperature or at 37°C for 10 minutes.

7. Washing: wash the specimen 3 times with 0.01M PBS, each time for 1-3 minutes.

8. Color development: Add 1-2 drops of freshly prepared DAB color development solution, 50 μL to 100 μL, and develop color at room temperature in the dark for 5 minutes to 10 minutes. Observe the staining results under an optical microscope. Do not develop too deep a color; rinse the slices with tap water to terminate color.

9. Counterstaining: add 100 μL of hematoxylin for counterstaining, incubate for about 5 minutes, wash with water for three times, use 1% hydrochloric acid ethanol for rapid fading treatment in 30 seconds, rinse with deionized water for 3 minutes, according to the intensity of the hematoxylin staining solution And the length of incubation time, contrast staining results lead to light blue to dark blue reaction of the nucleus, and overstaining or understaining may affect the judgment of the correct result.

10. Dehydration, transparency, sealing:

(1) Soak the slices in 70% alcohol for 2 minutes; then soak the slices in 80% alcohol for 2 minutes;

(2) Soak the slices in 90% alcohol for 2 minutes; then soak the slices in 95% alcohol for 2 minutes;

(3) Soak the slices in absolute ethanol for 2 minutes, replace the dyeing vat and repeat once; soak the slices in xylene for 2 minutes, replace the dyeing vat and repeat once;

(4) The slices were sealed with neutral resin glue, examined under a microscope, and filmed.

According to the above method, multi-antibody immunohistochemical detection was performed on normal liver tissue and HCC liver cancer tissue, and the results are shown in Figure 11 and Figure 12 .

Example 8

Embodiment 8 is compared with embodiment 7, and the difference between embodiment 8 and embodiment 7 is:

Preparation of Kit Standards:

Because the N-terminal fragment recombinant protein (NP_001158091.1, glypican-3isoform 4, 25-304AA) of the eukaryotic expression system GPC3 designed has the characteristics of high yield, high purity, and similar modification and folding to the natural protein, it is now used as a raw material. Standard buffer solution (the BSA that mass concentration is 3%, the NaCl that mass concentration is 0.6%, the Tween20 that mass concentration is 0.1%, the phosphate buffer solution that mass concentration is 0.05% Proclin-300, 0.2mol/L, pH is 7.0) were diluted to 0ng/ml, 0.125ng/ml, 0.5ng/ml, 2.5ng/ml, 10ng/ml, 50ng/ml respectively, prepared into standard products S1, S2, S3, S4, S5 and S6, used in GPC3 magnetic particle chemiluminescence kit (double antibody sandwich method) product, the results are as follows:

1. Standard curve

The standard products S1, S2, S3, S4, S5 and S6 were detected on the Beckman UniCel Dxl800 automatic chemiluminescence immunoassay analyzer with the GPC3 magnetic particle chemiluminescence kit, and the data are as follows:

Table 2 standard test data

Standard S1 S2 S3 S4 S5 S6 GPC3 concentration (ng/ml) 0 0.125 0.5 2.5 10 50 RLU 5156 42117 136525 683818 2651810 12856920

According to Table 2, draw a standard curve, see Figure 13.

2. Precision

The eukaryotic expression system GPC3 N-terminal fragment recombinant protein (NP_001158091.1, glypican-3isoform4, 25-304AA) was expressed and purified in three batches, and three batches of standard products were prepared respectively, which were recorded as batch 1, batch 2 and batch 3. Each batch of standard products was detected with GPC3 magnetic particle chemiluminescence kit on Beckman UniCel Dxl800 automatic chemiluminescence immunoassay analyzer, and the results are shown in Table 3.

Table 3 Precision test result table of different standard products

Standard batch one batch two batch three average cv S1 5076 6216 5123 5472 11.8% S2 42917 41665 42113 42232 1.5% S3 136521 135782 137564 136622 0.7% S4 682709 698785 682526 688007 1.4% S5 2692414 2715268 2610689 2672790 2.3% S6 12868600 11966632 13296451 12710561 5.3%

It can be seen from the above table that the precision: S1 test photon number CV≤20%, S2-S6 test photon number CV≤10%, meet the reagent requirements.

3. Stability:

The standard products S1, S2, S5 and S6 prepared in the same batch were divided into two groups, one group was subjected to simulated transportation at 42°C for 3 days, and the other group was stored at 4°C for 3 days. After the completion, the two groups of standard products were tested on the luminescence signal with the GPC3 magnetic particle chemiluminescence kit, and the luminescence value (42 degrees for 3 days)/luminescence value (4 degrees for 3 days) was the signal retention rate.

In the stability test of the standard product of the present invention, the detection data of the signal retention rate are shown in Table 4.

Table 4 The signal retention rate situation table of different standard products

It can be seen from Table 4 that the prepared standard substance has good stability, and the signal retention rate is above 90%.

One or more technical solutions in the embodiments of the present application also have at least the following technical effects or advantages:

(1) The coding gene provided in the embodiment of the present application, through the nucleotide sequence of the coding gene of the N-terminal fragment protein of the designed glypican 3, the mRNA formed in the transcription stage has no complicated secondary structure, The 5' end is relatively simple, which reduces the difficulty of the translation stage, improves translation efficiency, and thus increases the expression of the N-terminal fragment protein. Compared with the sequence of this patent, the nucleotide sequence of the N-terminal fragment of Glypican 3 used in the traditional design has 162 more bases at the 5' end, which may be complicated in structure and cause difficulties in the translation stage. Thereby affecting the yield of N-terminal fragment protein.

(2) The expression method of an N-terminal fragment protein provided in the embodiment of the present application can effectively carry out glycosylation modification and formation of disulfide bonds due to the adoption of a eukaryotic mammalian cell expression system, so it can be modified in the fold The phase is similar to that of natural GPC3, which can improve the accuracy of folding modification of the N-terminal fragment protein.

(3) The N-terminal fragment protein provided in the examples of this application is not easy to degrade, has good stability, and has the characteristics of relatively stable expression and purification process, while the traditionally prepared GPC3 full-length protein 25-559 has poor stability, and the purified protein has 70kDa, 40kDa and Three bands at 30 kDa.

(4) The N-terminal fragment protein provided in the examples of this application can be used as a standard raw material of the kit to achieve stable quantification to a certain extent.

(5) The method for preparing the N-terminal fragment protein provided in the examples of the present application can prepare the N-terminal protein of GPC3 with high yield, high purity, high activity and low cost.

It should be noted that in this article, relative terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply these No such actual relationship or order exists between entities or operations. Furthermore, the term "comprises", "comprises" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus comprising a set of elements includes not only those elements, but also includes elements not expressly listed. other elements of or also include elements inherent in such a process, method, article, or device. Without further limitations, an element defined by the statement "comprising..." does not exclude the presence of additional same elements in the process, method, article or device comprising said element.

Neither the endpoints nor any values of the ranges disclosed herein are limited to such precise ranges or values, and these ranges or values are understood to include values approaching these ranges or values. For numerical ranges, between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, these values Ranges should be considered as specifically disclosed herein.

The above descriptions are only specific embodiments of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Accordingly, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims (10)

1. The coding gene of the N-terminal fragment is characterized in that the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

2. An N-terminal fragment protein of glypican 3, wherein said N-terminal fragment protein is transcribed and translated from the coding gene of claim 1.

3. The coding gene according to claim 2, wherein the amino acid sequence of the N-terminal fragment protein is shown in SEQ ID NO. 2.

4. A recombinant vector comprising the coding gene of claim 1.

5. The recombinant vector of claim 4, wherein the recombinant vector comprises a eukaryotic vector.

6. An expression cell of a mammal, comprising the recombinant vector of claim 4 or 5.

7. A method for preparing an N-terminal fragment protein, comprising:

synthesizing the coding gene of claim 1;

constructing the recombinant vector according to claim 4 or 5 with the coding gene;

transfecting competent cells by the recombinant vector, and culturing to obtain an expression product of the N-terminal fragment;

and separating the expression product, and purifying to obtain the N-terminal fragment protein.

8. A polyclonal antibody of an N-terminal fragment protein, wherein the polyclonal antibody specifically binds to the N-terminal fragment protein of claim 2 or 3.

9. A method of making the polyclonal antibody of claim 8, wherein the method comprises:

immunizing an animal model with the N-terminal fragment protein of claim 2 or 3 to obtain a primary polyclonal antibody;

and carrying out antigen affinity purification on the primary polyclonal antibody to obtain the specific polyclonal antibody.

10. Use of an N-terminal fragment of glypican 3, said use comprising:

use of the N-terminal fragment protein of claim 2 or 3 in the preparation of a kit.

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