Collagen type iv recombinant antigen and use thereof

Abstract

The embodiment of the present application discloses a type IV collagen recombinant antigen and application. From N terminal to C terminal, the amino acid sequence fragments shown as SEQ ID NO. 1, the amino acid sequence fragments shown as SEQ ID NO. 2 and the amino acid sequence fragments shown as SEQ ID NO. 3 are sequentially spliced. The multi-subunit fragments α2 (7S), α3 (724-778) and α5 (NC1) are recombined in the present application, so that the recombinant IVC antigen with considerable expression amount and stability can be obtained, the antibody affinity after immunization can be increased, and the IVC antibody with higher titer can be obtained.

Description

TECHNICAL FIELD

[0001] The embodiment of the present application relates to the field of bioengineering technology, and particularly relates to a type IV collagen recombinant antigen and application. BACKGROUND

[0002] Basement membranes are multifunctional, providing structural support and polarity to epithelia and attaching them to underlying connective tissue, stroma or interstitium. Basement membranes control epithelial growth and differentiation during embryogenesis and organogenesis and mark the path of cell migration and epithelialization during tissue repair. In cancer, basement membranes form a barrier to tumor cell invasion, and disruption of basement membranes promotes cancer metastasis. Basement membranes carry a net negative charge and act as a charge-selective filtration barrier to regulate the passage of negatively charged molecules from the bloodstream to tissues. This is notable in the kidney filtration barrier. In the lung, basement membranes form part of the blood-air barrier. In the central nervous system, the basal lamina of capillaries contributes to the blood-brain barrier. Vascular basement membranes show anti-angiogenic activity. Basement membranes bind growth factors, thereby regulating their availability to cells and tissues. Basement membranes are composed mainly of type IV collagen (IVC), laminin, nidogen and perlecan.

[0003] IVC is widely distributed in basement membranes, and its presence in basement membranes was first demonstrated by immunological methods and X-ray diffraction. IVC is a non-fibrillar collagen, classified as network / lattice / sheet collagen. In mammals, there are six genetically distinct IVC chains, alpha1(IV)-alpha6(IV), encoded by COL4A1-COL4A6 genes, and all have a similar domain structure. Each alpha chain is ~400 nm long and can be divided into three domains: a short N-terminal non-collagenous 7S domain (26 kDa, ~28 nm in length), a long middle triple-helical collagen domain (120 kDa in length, ~320 nm or ~1400 amino acids), and a C-terminal globular non-collagenous domain NC1 (25 kDa, ~52 nm or ~230 amino acids in length). Uniquely, the central helical portion of the alpha chain containing 1400 Gly-X-Y sequences is interrupted by about 20 short non-collagenous regions, which provide flexibility to the molecule and thus to the basement membrane. The 7S domain gets its name from its sedimentation properties when centrifuged after proteolytic digestion of IVC. Both the 7S and NC1 domains are involved in the formation of IVC networks.

[0004] With regard to IVC gene expression, the alpha1(IV)2alpha2(IV) isoform is the predominant isoform and is ubiquitous in basement membranes of all epithelial tissues. It is the only form found in the liver. The alpha3(IV)alpha4(IV)alpha5(IV) forms are present in the kidney, lung and neuromuscular junction. The alpha5(IV)2alpha6(IV) forms are present in smooth muscle and neuromuscular junctions.

[0005] In human liver, IVC expression is upregulated, cirrhosis increases 14-fold, which is the highest relative increase among all collagen types. In hepatitis C-related fibrosis, IVC immunoreactivity rises sharply with changes in the progression of fibrosis, and its expression is a useful discriminator between early and late stages of fibrosis. Antigens associated with the 7S and NC1 domains of IVC have been evaluated as non-invasive biomarkers of chronic liver disease. The increased expression of IVC and the deposition of laminin in the Disse space lead to the formation of perisinusoidal basement membranes in liver fibrosis.

[0006] Alport syndrome (AS) is a genetic kidney disease caused by genetic variations in COL4A3, COL4A4 and COL4A5 genes, of which COL4A5 accounts for 80%. These genes encode proteins that form a heterodimer α3α4α5, which constitutes type IV collagen, the main component of GBM. Mutations in these genes affect the synthesis, assembly, deposition or function of type IV collagen molecules in podocytes in the kidney (or a combination of these genes). Dysfunctional GBM leads to hematuria and albuminuria, leading to chronic inflammation and fibrosis.

[0007] Type IV collagen is also present in the cochlea (stria vascularis), cornea (Descemet and Bowamn membranes), lens capsule and retina (internal limiting membrane and Bruch's membrane). Extrarenal manifestations can also occur, such as sensorineural hearing loss and ocular abnormalities (corneal opacity, anterior lens, punctate retinopathy, temporal retinal thinning, and more rarely, posterior polymorphous corneal dystrophy, macular hole and maculopathy). Rare manifestations include leiomyomas of the esophagus, tracheobronchial tree and female genitalia, and are associated with contiguous gene deletions in COL4A6 and COL4A5. Vascular abnormalities such as aortic aneurysm have also been reported.

[0008] The conventional IVC antibody is a monoclonal antibody obtained by immunizing mice with all genes expressed by COL4A1-COL4A6 or 7S+NC1 domains. Since it is a single subunit, the content in the serum is not accurate in the later detection reagent configuration. Therefore, a new antigen is needed to be immunized to obtain an antibody with better specificity and better detection sensitivity. SUMMARY

[0009] To this end, the embodiments of the present application provide a type IV collagen recombinant antigen and an application thereof.

[0010] In order to achieve the above object, the embodiments of the present application provide the following technical solutions:

[0011] According to a first aspect of the embodiments of the present application, the present application provides a collagen type IV recombinant antigen comprising, from N-terminal to C-terminal, sequentially spliced amino acid sequence fragments as shown in SEQ ID NO. 1, as shown in SEQ ID NO. 2, and as shown in SEQ ID NO. 3.

[0012] Further, the N-terminal of the recombinant antigen is connected with a signal peptide sequence.

[0013] Further, the signal peptide sequence is a human chymotrypsinogen signal peptide sequence as shown in SEQ. ID NO. 4.

[0014] Further, the C-terminal of the recombinant antigen is connected with a protein tag.

[0015] Further, the protein tag is a his tag.

[0016] According to a second aspect of the embodiments of the present application, the present application provides a nucleic acid encoding the collagen type IV recombinant antigen as described in any one of the above.

[0017] According to a third aspect of the embodiments of the present application, the present application provides an expression vector comprising the nucleic acid as described above.

[0018] According to a fourth aspect of the embodiments of the present application, the present application provides a host cell comprising the expression vector as described above.

[0019] According to a fifth aspect of the embodiments of the present application, the present application provides use of the collagen type IV recombinant antigen as described in any one of the above, the nucleic acid as described above, the expression vector as described above, or the host cell as described above in the preparation of a kit for detecting the content of collagen type IV in serum.

[0020] According to a sixth aspect of the embodiments of the present application, the present application provides a kit comprising the collagen type IV recombinant antigen as described in any one of the above.

[0021] The embodiments of the present application have the following advantages:

[0022] The present application recombines the multi-subunit fragments α2 (7S), α3 (724-778) and α5 (NC1), and can obtain a recombinant IVC antigen with considerable expression amount and stability, and can increase the antibody affinity after immunization, and obtain an IVC antibody with higher titer. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to make the technical solutions of the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the description of the embodiments or the prior art will be briefly introduced. Obviously, the accompanying drawings in the following description are only exemplary and, for those skilled in the art, other drawings can be obtained from the provided drawings without any creative effort.

[0024] Figure 1 SDS-PAGE verification results of the recombinant protein IVC-R of the present application. DETAILED DESCRIPTION

[0025] The embodiments of the present application are illustrated by specific examples below, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the description. Obviously, the described examples are part of the embodiments of the present application, but not all the embodiments. Based on the examples in the present application, all other embodiments obtained by those skilled in the art without any creative effort are within the scope of protection of the present application. In the following examples, the used biochemical reagents are commercially available unless otherwise specified, and all the methods are conventional methods.

[0026] Example 1

[0027] Preparation of type IV collagen recombinant antigen (named IVC-R)

[0028] The signal peptide sequence (SEQ ID NO. 4), α2 (7S) (SEQ ID NO. 1), α3 (724-778) (SEQ ID NO. 2), α5 (NC1) (SEQ ID NO. 3) and his protein tag are sequentially spliced from N-terminal to C-terminal in a tandem manner, and the amino acid sequence is:

[0029] MAFLWLLSCWALLGTTFGFLAQSVLAGVKKFDVPCGGRDCSGGCQCYPEKGGRGQPGPVGPQGYNGPPGLQGFPGLQGRKGDKGERGAPGVTGPKGDVGARGVSGFGKMGEPGLPGKPGLPGAKGEPAVAMPGGPGTPGFPGERGNSGEHGEIGLPGLPGLGFLITRHSQTTDAPQCPQGTLQVYEGFSLLYVQGNKRAHGQDLGTAGSCLRRFSTMPFMFCNINNVCNFASRNDYSYWLSTPEPMPMSMQPLKGQSIQPFISRCAVCEAPAVVIAVHSQTIQIPHCPQGWDSLWIGYSFMMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNYYANSYSFWLATVDVSDMFSKPQSETLKAGDLRTRISRCQVCMKRTHHHHHH (SEQ ID NO. 5).

[0030] The gene sequence encoding the recombinant protein IVC-R described above was constructed according to the conventional method in the art, synthesized into pcDNA3.1 vector, and a recombinant expression vector pcDNA3.1-IVC-R was constructed (completed by Shenguo Bioengineering (Shanghai) Co., Ltd.). The recombinant protein IVC-R was obtained by transfecting 293T cells, expressing, and purifying. The process is as follows:

[0031] (1) Prepare two 3 cm culture dishes, add 10 ml transfection buffer (Opti-MEM, 101000046) and 200 μg sterile plasmid DNA to one of the culture dishes, and mix gently by blowing; add 10 ml transfection buffer and 0.5 ml transfection reagent (Polysciences, 26008-50) to the other culture dish, and mix gently by blowing;

[0032] (2) Add all the liquid in the culture dish containing the transfection reagent dropwise to the culture dish containing the plasmid, shake while adding, and mix gently by blowing;

[0033] (3) Stand at room temperature for 30 minutes to prepare the plasmid-vector complex;

[0034] (4) Take 293T cells (density 6 x 10 6 cells / ml) from the constant temperature shaker, add the prepared plasmid-vector complex dropwise while shaking, and place in a carbon dioxide constant temperature shaker for culture;

[0035] (5) Collect the cells at 7th day after transfection, centrifuge at 9000 rpm for 15 min, collect the supernatant;

[0036] (6) Purify the supernatant by nickel column: 5 ml column, peristaltic pump speed 7.5 rpm / min, 1 x PBS 50 mM imidazole solution to remove impurities, 1 x PBS 300 mM imidazole solution elution.

[0037] Purified protein was verified by SDS-PAGE, the results are shown in Figure 1 .

[0038] Example 2

[0039] The purified recombinant protein IVC-R of Example 1 was used to immunize SPF level purebred BALB / C mice, and α2(7S)-α5(NC1) (hereinafter referred to as IVC-S, which is only different from IVC-R in that it does not contain the α3(724-778) subunit fragment) was used as a control.

[0040] I. Animal immunization

[0041] (1) Primary immunization, the recombinant protein was mixed with Freund's complete adjuvant at a ratio of 1:1 to emulsify, and the mice were subcutaneously injected, with an immunization dose of 50 μg of recombinant protein per mouse, and the interval was 2 weeks;

[0042] (2) Secondary immunization, the dose was the same as above, and Freund's incomplete adjuvant was used instead of Freund's complete adjuvant, with an interval of 2 weeks;

[0043] (3) Third immunization, the dose was the same as above, without adjuvant, intraperitoneal injection, blood was collected 7 days later to measure the titer and detect the immune effect, with an interval of 2 weeks, and spleen cells were taken.

[0044] II. Cell fusion

[0045] (1) Mix 1 x 10 8 splenocytes with 1 x 10 7 myeloma cells in a 50 ml fusion tube, add incomplete medium (Paisi Luo, PM150210) to 30 ml, mix well, centrifuge at 1000 r / min for 5 minutes, and aspirate the supernatant as much as possible;

[0046] (2) Add 1 ml of 50% PEG preheated at 37°C with a pipette within 30 seconds, stirring gently while adding, and aspirate the pipette for 2 minutes;

[0047] (3) Add preheated incomplete culture medium to terminate PEG action, and add 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml successively within 2 minutes, centrifuge at 1000 r / min for 5 minutes, and discard the supernatant.

[0048] (4) Add 5ml complete medium (Paisi Luo, PM150210A), gently blow the cells to suspend and mix, then add complete medium to 40-50ml. Distribute 96-well cell culture plates, 100ul per well, then place the culture plates in a 37℃, 5% CO2 incubator for culture;

[0049] (5) After 6h, add selection medium (Paisi Luo, PM150211) to each well, 50ul, and change half of the selection medium after 3 days;

[0050] (6) Frequently observe the growth of hybridoma cells, and when the cells grow to cover more than 1 / 10 of the bottom area of the well after fusion, aspirate the supernatant for antibody detection.

[0051] III. Antibody titer detection

[0052] (1) Antigens IVC-R and IVC-S are diluted to 10ug / ml with coating solution (0.05M carbonate buffer, self-made);

[0053] (2) Add 100ul / well to the enzyme-labeled plate wells, repeat twice, and place at 37℃ for 2h;

[0054] (3) Discard the liquid in the wells, and wash with washing solution (0.01M PBS, self-made) for 3 times, 3min each time, and pat dry;

[0055] (4) Add 100ul blocking solution (2% BSA, Beijing Zhulong Medical Biology) per well, and block at 37℃ for 1h;

[0056] (5) Wash with washing solution for 3 times, 3min each time, and pat dry;

[0057] (6) Add 100ul of the corresponding hybridoma cell culture supernatant per well, and set up positive IVC antibody (COL4A3 full-length immunized antigen-derived antibody), negative control and blank control at the same time, incubate at 37℃ for 30min, wash and pat dry;

[0058] (7) Add enzyme-labeled secondary antibody, 100ul per well, incubate at 37℃ for 30min, wash and pat dry;

[0059] (8) Add substrate solution, 10ul of freshly prepared substrate solution per well, and incubate at 37℃ for 20min;

[0060] (9) Terminate the reaction with 2mol / L H2SO4, and read the OD value on the enzyme-linked immunosorbent reader;

[0061] (10) Result determination: P / N≧2.1, or P≧N+3SD is positive. If the negative control well is colorless or close to colorless, and the positive control well is clearly colored, the result can be observed directly with the naked eye.

[0062] Table 1 Statistics of antibody detection results

[0063] Antibody Positive IVC antibody IVC-R antibody IVC-S antibody Negative control Blank control Test 1 0.813 0.905 0.624 0.056 0.040 Test 2 0.806 0.908 0.621 0.057 0.040 Mean 0.810 0.907 0.623 0.057 0.040

[0064] The results show that the titer of the IVC-R antibody is higher than that of the antibodies obtained by immunization with IVC full-length and IVC-S, and the difference is extremely significant (P < 0.001). It is shown that the constructed fusion recombinant protein plus the helical structure α3 (724-778) conferring flexibility to the basement membrane has the effect of increasing IVC binding.

[0065] Example 3

[0066] The IVC-R antibody screened is applied to the preparation of a latex kit, and details are shown in Table 2.

[0067] Table 2 Main components of the IVC-R antibody latex detection kit

[0068]

[0069]

[0070] In order to verify that the detection value of the kit produced by the present application is more accurate, a quality control antigen is detected.

[0071] Detection method:

[0072] 2 μL of the sample to be tested is taken, 200 μL of reagent R1 is added, and incubation is carried out at 37°C for 5 min;

[0073] 40 μL of latex reagent R2 is added, incubation is carried out at 37°C for 30 s, and the absorbance A1 is read;

[0074] Incubation is continued at 37°C for 5 min, and the absorbance A2 is read;

[0075] ΔA = A2-A1 is calculated, and the content of IVC in the sample to be tested is calculated according to ΔA.

[0076] The sample content is 450 ng / ml, and 10 times of detection are carried out compared with a conventional detection reagent, and the detection results are shown in Table 3.

[0077] Table 3 IVC-R antibody detection results

[0078] Number of tests IVC-R reagent (ng / ml) Routine IVC test reagent (ng / ml) 1 450 449.7 2 452.4 450.1 3 451.8 441.2 4 450.2 443.9 5 453.7 432 6 455.2 439.2 7 452.1 441.3 8 450.7 438.9 9 451.3 445.6 10 452.5 436.8 Mean 451.99 441.87 SD 1.52 5.36 CV 0.34％ 1.21％

[0079] As can be seen from Table 3, the detection CV of the two kits is within a reasonable range, but the detection result selected by IVC-R is closer to the reagent value of the quality control. It is shown that the antibody titer obtained after immunization with the antigen expressed by multi-fragment recombination provided by the present application is high, and the reagent detection is more reliable.

[0080] While the application has been described in detail with particular references to specific embodiments thereof, it will be understood by those skilled in the art that various modifications or changes in form and details can be made therein without departing from the spirit and scope thereof. It is therefore intended that the application be construed as including all such modifications and changes as fall within the scope of the appended claims.

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Claims

1. A recombinant antigen of collagen type IV, characterized in that, The amino acid sequence of the collagen type IV recombinant antigen is shown in SEQ ID NO.

5.

2. A nucleic acid encoding the collagen type IV recombinant antigen of claim 1.

3. An expression vector, characterized by, comprising the nucleic acid of claim 2.

4. A host cell, characterized in that, comprising the expression vector of claim 3.

5. Use of the collagen type IV recombinant antigen of claim 1, the nucleic acid of claim 2, the expression vector of claim 3, the host cell of claim 4 in the preparation of a kit for detecting the collagen type IV content in serum.

6. A kit characterized in that, comprising the collagen type IV recombinant antigen of claim 1.

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