CN109678950A - Spink1 antigen, the antibody that spink1 can be specifically bound and its function fragment and its application and product - Google Patents

Spink1 antigen, the antibody that spink1 can be specifically bound and its function fragment and its application and product Download PDF

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CN109678950A
CN109678950A CN201910014180.1A CN201910014180A CN109678950A CN 109678950 A CN109678950 A CN 109678950A CN 201910014180 A CN201910014180 A CN 201910014180A CN 109678950 A CN109678950 A CN 109678950A
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antibody
spink1
seq
function fragment
2cdr
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CN109678950B (en
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荆东辉
陈丽丽
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Tianjin Reasonbio Co ltd
Haoling Cell Technologies Corp
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Tianjin Reasonbio Co ltd
Haoling Cell Technologies Corp
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    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
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    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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Abstract

The present invention relates to field of biotechnology, specifically, providing spink1 antigen, the antibody that can specifically bind spink1 and its function fragment and its application and product.Spink1 antigen provided by the invention, comprising: SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 and the former any one derivative polypeptide have good immunogenicity.Above-mentioned antigen respectively obtains first antibody, secondary antibody and third antibody, and obtains the hybridoma cell strain that can produce above-mentioned three kinds of antibody respectively, and the potency of antibody is high, and sensitivity is good, stable structure；The antibody and its function fragment of the spink1 of high quality can be stablized, be efficiently produced to hybridoma, can be used as engineered strain and carry out industrialized production.The present invention also provides the application of above-mentioned antibody and its function fragment and the product using it.

Description

Spink1 antigen, the antibody that spink1 can be specifically bound and its function fragment and It is applied and product
Technical field
The present invention relates to field of biotechnology, in particular to spink1 antigen, can specifically bind spink1's Antibody and its function fragment and its application and product.
Background technique
Serpin (serine protease inhibitors, SERPIN) is to be distributed most wide, scale Maximum protease inhibitors superfamily, serpin kazal1 type (serine peptidase Inhibitor Kazal type 1, SPINK1) as a member in the family, it has received widespread attention in recent years, it is A kind of secreted polypeptide being made of 56 amino acid residues, most important function are to inhibit the activity of trypsase.
Studies have shown that SPINK1 is a potential cancer biomarker in vivo, it is equal in a plurality of types of cancers There is expression, can be used to identify the risk of patient and the poor prognosis of tumour.It is thin that SPINK1 high is expressed in kinds of tumors Born of the same parents, including liver cancer, oophoroma, cancer of pancreas, prostate cancer, breast cancer, colorectal cancer, bladder cancer, gastric cancer or kidney etc..It is right SPINK1, which carries out detection and research, has great importance to the Regulation Mechanism of kinds of tumors and early diagnosis.
Currently, the research of SPINK1 is concentrated mainly in relative expression level, Regulation Mechanism and nucleic acid level detection, and For the field of SPINK1 Protein Detection, mostly in the stage explored, effect is not satisfactory, and Effective Antigens are still indefinite, The product for the antibody for being used to detect SPINK1 albumen simultaneously is seldom, and there are lower (the minimum works of at high cost, potency for these products Be 0.8ug/ml with concentration) defect.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of spink1 antigen, to alleviate in the prior art for spink1 albumen Detection the technical issues of lacking Effective Antigens.
The second object of the present invention is to provide a kind of antibody and its function fragment that can specifically bind spink1, with Alleviate the technical problem that spink1 antibody is at high cost, potency is low in the prior art.
The third object of the present invention is to provide the antibody and its functional sheet that a kind of generation can specifically bind spink1 The antibody and its functional sheet of the spink1 of high quality can be stablized, be efficiently produced to the hybridoma of section, the hybridoma Section can be used as engineered strain and carry out industrialized production.
The fourth object of the present invention is to provide a kind of isolated nucleic acid molecules and carrier, efficiently quickly to express To the antibody and its function fragment of spink1.
The fifth object of the present invention is to provide the application of above-mentioned spink1 antigen or antibody and its function fragment
The sixth object of the present invention is to provide a kind of composition and kit, with alleviate lack in the prior art with Spink1 is the diagnosis or treatment product of target.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of spink1 antigen is following a)-f) in any polypeptide:
a)RQTSILIQKSGPC(SEQ ID NO.1)；
B) amino acid sequence shown in SEQ ID NO.1 by the substitution of one or several amino acid residues and/or is lacked Lose and/or add and have the polypeptide as derived from a) of spink1 epitope function；
c)MKVTGIFLLSALALLSLS(SEQ ID NO.2)；
D) amino acid sequence shown in SEQ ID NO.2 by the substitution of one or several amino acid residues and/or is lacked Lose and/or add and have the polypeptide as derived from c) of spink1 epitope function；
e)GNTGADSLGREAKCYNE(SEQ ID NO.3)；
F) amino acid sequence shown in SEQ ID NO.3 by the substitution of one or several amino acid residues and/or is lacked Lose and/or add and have the polypeptide as derived from e) of spink1 epitope function.
A kind of antibody and its function fragment that can specifically bind spink1, is following a1)-c1) in it is any anti- Body and its function fragment:
A1) the first antibody of SEQ ID NO.1, the first antibody and its function fragment include light chain and heavy chain；
The light chain has the light chain CDR being made of 1CDR-L1,1CDR-L2,1CDR-L3；The heavy chain have by The heavy chain CDR of 1CDR-H1,1CDR-H2,1CDR-H3 composition；
The amino acid sequence of described 1CDR-L1,1CDR-L2,1CDR-L3 respectively as shown in SEQ ID NO.4,5 and 6, The amino acid sequence of described 1CDR-H1,1CDR-H2,1CDR-H3 are respectively as shown in SEQ ID NO.7,8 and 9；
B1) the secondary antibody of SEQ ID NO.2, the secondary antibody and its function fragment include light chain and heavy chain；
The light chain has the light chain CDR being made of 2CDR-L1,2CDR-L2,2CDR-L3；The heavy chain have by The heavy chain CDR of 2CDR-H1,2CDR-H2,2CDR-H3 composition；
The amino acid sequence of described 2CDR-L1,2CDR-L2,2CDR-L3 are respectively such as the institute of SEQ ID NO.10,11 and 12 Show, the amino acid sequence of described 2CDR-H1,2CDR-H2,2CDR-H3 are respectively as shown in SEQ ID NO.13,14 and 15；
C1) the third antibody of SEQ ID NO.2, the third antibody and its function fragment include light chain and heavy chain；
The light chain has the light chain CDR being made of 3CDR-L1,3CDR-L2,3CDR-L3；The heavy chain have by The heavy chain CDR of 3CDR-H1,3CDR-H2,3CDR-H3 composition；
The amino acid sequence of described 3CDR-L1,3CDR-L2,3CDR-L3 are respectively such as the institute of SEQ ID NO.16,17 and 18 Show, the amino acid sequence of described 3CDR-H1,3CDR-H2,3CDR-H3 are respectively as shown in SEQ ID NO.19,20 and 21.
Further, the antibody and its function fragment include spink1 chimeric antibody and its function fragment；
Preferably, the antibody include ox, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, Donkey, deer, ermine, chicken, duck, goose, turkey, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD of cockfighting or people are any wherein One of constant region sequence；
Preferably, the function fragment includes F (ab ')2, Fab ', Fab, Fv, scFv, bispecific antibody and antibody it is minimum One of recognition unit is a variety of.
A kind of hybridoma generating the above-mentioned antibody that can specifically bind spink1 and its function fragment, is as follows A2)-c2) in any hybridoma:
A2 the hybridoma of first antibody) is generated, deposit number is CGMCC No.15196；
B2 the hybridoma of secondary antibody) is generated, deposit number is CGMCC No.15698；
C2 the hybridoma of third antibody) is generated, deposit number is CGMCC No.15699.
A kind of isolated nucleic acid molecules, the nucleic acid molecules are selected from following nucleic acid:
A3), DNA or RNA encodes above-mentioned antibody and its function fragment；
B3 the nucleic acid of complementary nucleic acid defined in) and a3).
A kind of carrier, including above-mentioned nucleic acid molecules.
Above-mentioned spink1 antigen is in following a4) or b4) in application:
A4) preparation can specifically bind the antibody of spink1；
B4) screening is using spink1 albumen as the drug of target spot.
Above-mentioned antibody and its function fragment are in following a5)-d5) in application:
A5), for detecting the expression of spink1 albumen；
B5), as using spink1 albumen as the carrier of the drug of target spot；
C5), for purifying or separating spink1 albumen；
D5), it is used to prepare the product of detection spink1 albumen；
Preferably, the source of the spink1 albumen be ox, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, Rabbit, camel, donkey, deer, ermine, chicken, duck, goose, turkey, cockfighting or people；
Preferably, the a5) in detection method include western blot method, immunohistochemistry, immunostaining or ELISA method；
Preferably, the a5) in the sample of detection include whole blood, serum, blood plasma, urine or tumour vesica；
Preferably, the b5) in drug it is straight for treating liver cancer, oophoroma, cancer of pancreas, prostate cancer, breast cancer, knot Intestinal cancer, bladder cancer, gastric cancer or kidney；
Preferably, the d5) in product include ELISA kit, colloidal gold kit or paramagnetic particle method kit.
A kind of composition, the composition include above-mentioned antibody and its function fragment and at least one diagnosticum and/or control Treat agent；
The antibody and its function fragment and diagnosticum and/or therapeutic agent to form immune conjugate to be coupled.
A kind of kit containing above-mentioned antibody and its function fragment, the kit are ELISA kit, colloidal gold examination Agent box or paramagnetic particle method kit, the kit is for detecting spink1 albumen.
Compared with prior art, the invention has the benefit that
Spink1 antigen provided by the invention has strong antigen activity, select peptide chain and non-recombinant protein as antigen more Added with locking antigenic determinant is helped, more theories integrations are provided for subsequent ELISA pairing and antibody drug research.SEQ ID NO.1 is the aa67-79 section of spink1 albumen, which is high hydrophilic region, can produce the antibody of high-titer；SEQ ID NO.2 is the aa1-18 section of spink1 albumen, which can increase the specificity of identification liver cancer cells；SEQ ID NO.3 For the aa19-35 section of spink1 albumen, which is more easily exposed to the outside of spink1 albumen, in ELAISA double antibodies sandwich It is easier to be formed with C-terminal antibody in method complementary.Spink1 antigen provided by the invention, antigen active is good, is used to prepare spink1 egg Bai Kangti or screening are efficient as the drug of target spot, accurate using spink1 albumen, have important industrial application value.
Antibody and its function fragment provided by the invention can specifically bind spink1 albumen, and potency is high, and sensitivity is good, Stable structure can be applied to detection, identification, separation and the purifying of spink1 albumen, also can be used as using spink1 albumen as mesh The carrier of target point.
Hybridoma provided by the invention can generate antibody and its function fragment with above-mentioned CDR sequence, this is miscellaneous It hands over oncocyte that can stablize, efficiently produce the antibody and its function fragment of spink1 of high quality, can be used as engineered strain Carry out industrialized production.
Nucleic acid molecules provided by the invention, can with encoding such antibodies and its function fragment or with the complementary nucleic acid of coding, The nucleic acid molecules can be applied to genetic engineering, construct carrier or host cell containing the nucleic acid molecules, so as to efficiently quick Expression obtain the antibody and its function fragment of spink1.
Composition provided by the invention is with above-mentioned antibody and its function fragment and diagnosticum and/or the coupled shape of therapeutic agent At immune conjugate.For the identifying of spink1 albumen, purify, detect, separate or using spink1 as the research of target, Drug etc..Since the antibody and its high specific and potency of function fragment, the composition can be applied to various fields and field Scape.
Kit provided by the invention, kit include but are not limited to ELISA kit, colloidal gold kit or paramagnetic particle method Kit, for detecting spink1 albumen, high sensitivity is at low cost, as a result accurately.
Detailed description of the invention
It, below will be to tool in order to illustrate more clearly of this hair invention specific embodiment or technical solution in the prior art Body embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing be some embodiments of the present invention, for those of ordinary skill in the art, what is do not made the creative labor Under the premise of, it is also possible to obtain other drawings based on these drawings.
Fig. 1 is first antibody SDS-PAGE testing result figure in the embodiment of the present invention 8, wherein M:maker, 1 and 2: the One antibody；
Fig. 2 is secondary antibody SDS-PAGE testing result figure in the embodiment of the present invention 9；
Fig. 3 is third antibody SDS-PAGE testing result figure in the embodiment of the present invention 10, wherein M:maker, 1: third Antibody；
Fig. 4 is first antibody western blot testing result figure in the embodiment of the present invention 11, wherein 1-79: liver cancer is thin Born of the same parents are the spink1 holoprotein (theoretical molecular weight 8.5KD) of expression, 24-79: the spink1 holoprotein (band of plant cell expression There are GST label, theoretical molecular weight 33KD)；
Fig. 5 is third antibody western blot testing result figure in the embodiment of the present invention 12, wherein 1: liver cancer cells table The spink1 holoprotein reached；2: blank control.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.
A kind of spink1 antigen is following a)-f) in any polypeptide:
a)RQTSILIQKSGPC(SEQ ID NO.1)；
B) amino acid sequence shown in SEQ ID NO.1 by the substitution of one or several amino acid residues and/or is lacked Lose and/or add and have the polypeptide as derived from a) of spink1 epitope function；
c)MKVTGIFLLSALALLSLS(SEQ ID NO.2)；
D) amino acid sequence shown in SEQ ID NO.2 by the substitution of one or several amino acid residues and/or is lacked Lose and/or add and have the polypeptide as derived from c) of spink1 epitope function；
e)GNTGADSLGREAKCYNE(SEQ ID NO.3)；
F) amino acid sequence shown in SEQ ID NO.3 by the substitution of one or several amino acid residues and/or is lacked Lose and/or add and have the polypeptide as derived from e) of spink1 epitope function.
Spink1 antigen provided by the invention has good immunogenicity, select peptide chain and non-recombinant protein as resisting Original more facilitates to lock antigenic determinant, provides more theories integrations for subsequent ELISA pairing and antibody drug research.SEQ ID NO.1 is the aa67-79 section of spink1 albumen, the polypeptide chain and be high hydrophilic region, and the antigen and its derivative antigen can be with Generate the antibody of high-titer；SEQ ID NO.2 is the aa1-18 section of spink1 albumen, the expression point of spink1 in liver cancer cells Son amount (about 10kd) is greater than expected molecular weight (about 6.3kd), should be the result shows that the change of molecular weight may be due to liver cancer cells Middle N-terminal signal peptide peptide chain is not caused by complete excision, therefore the polypeptide chain and its derivative antigen can increase identification liver cancer cells Specificity；SEQ ID NO.3 is the aa19-35 section of spink1 albumen, which is more easily exposed to the outer of spink1 albumen Side is easier to be formed with C-terminal antibody in ELAISA double-antibody method complementary.Spink1 antigen provided by the invention, antigen active It is good, it is economical, effective, new think of is provided as the drug of target spot to prepare spink1 protein antibodies or screening using spink1 albumen Road.The specific humoral immune response for generating and being directed to spink1 albumen, obtained antibody can be induced in mouse model Potency is high, high-quality.
A kind of antibody and its function fragment that can specifically bind spink1, is following a1)-c1) in it is any anti- Body and its function fragment:
A1) the first antibody of SEQ ID NO.1, first antibody and its function fragment include light chain and heavy chain；
Light chain has the light chain CDR being made of 1CDR-L1,1CDR-L2,1CDR-L3；Heavy chain have by 1CDR-H1, The heavy chain CDR of 1CDR-H2,1CDR-H3 composition；
The amino acid sequence of 1CDR-L1,1CDR-L2,1CDR-L3 respectively as shown in SEQ ID NO.4,5 and 6, The amino acid sequence of 1CDR-H1,1CDR-H2,1CDR-H3 are respectively as shown in SEQ ID NO.7,8 and 9；
B1) the secondary antibody of SEQ ID NO.2, secondary antibody and its function fragment include light chain and heavy chain；
Light chain has the light chain CDR being made of 2CDR-L1,2CDR-L2,2CDR-L3；Heavy chain have by 2CDR-H1, The heavy chain CDR of 2CDR-H2,2CDR-H3 composition；
The amino acid sequence of 2CDR-L1,2CDR-L2,2CDR-L3 respectively as shown in SEQ ID NO.10,11 and 12, The amino acid sequence of 2CDR-H1,2CDR-H2,2CDR-H3 are respectively as shown in SEQ ID NO.13,14 and 15；
C1) the third antibody of SEQ ID NO.2, third antibody and its function fragment include light chain and heavy chain；
Light chain has the light chain CDR being made of 3CDR-L1,3CDR-L2,3CDR-L3；Heavy chain have by 3CDR-H1, The heavy chain CDR of 3CDR-H2,3CDR-H3 composition；
The amino acid sequence of 3CDR-L1,3CDR-L2,3CDR-L3 respectively as shown in SEQ ID NO.16,17 and 18, The amino acid sequence of 3CDR-H1,3CDR-H2,3CDR-H3 are respectively as shown in SEQ ID NO.19,20 and 21.
It is known in the art that the binding specificity and affinity of antibody are mainly determined by CDR sequence, according to mature, known Existing every technology the amino acid sequence in non-CDR region domain can be changed easily and obtaining has similar bioactivity Variant.
Antibody and its function fragment provided by the invention can specifically combine spink1 albumen, and potency is high, sensitivity Good, stable structure can be applied to detection, identification, separation and the purifying of spink1 albumen, also can be used as and is with spink1 albumen The carrier of target.
In certain embodiments of the present invention, antibody and its function fragment include spink1 chimeric antibody and its functional sheet Section.
In certain embodiments of the present invention, antibody include ox, horse, cow, pig, sheep, goat, rat, mouse, Dog, cat, rabbit, camel, donkey, deer, ermine, chicken, duck, goose, turkey, the IgG 1 of cockfighting or people, IgG 2, IgG 3, IgG 4, IgA, The sequence of one of them any constant region of IgM, IgE or IgD.
In certain embodiments of the present invention, function fragment includes F (ab ')2, it is Fab ', Fab, Fv, scFv, double special One of antibody and antibody atom are a variety of.
" function fragment " of the present invention particularly refers to for spink1 with anti-with maternal antibody phase homospecificity Body segment.It further include half-life period increased any segment in addition to above-mentioned function fragment.
ScFv (sc=is single-stranded), bispecific antibody (diabodies).
These function fragments usually have binding specificity identical with its derived antibodies.Those skilled in the art are according to this The content recorded in invention description infers, antibody fragment of the invention can pass through the method for such as enzymic digestion (including stomach cardia Enzyme or papain) and/or by electronation divide disulfide bond method obtain above-mentioned function fragment.
Antibody fragment can also by be also genetic recombination technology known to those skilled in the art or for example, by from The automatic peptide synthesizer of the sale such as dynamic peptide synthesizer, such as Applied BioSystems, is obtained by peptide synthesis.
It is a kind of generate it is above-mentioned can specifically in conjunction with spink1 antibody and its function fragment hybridoma, be as Lower a2)-c2) in any hybridoma:
A2 the hybridoma of first antibody) is generated, deposit number is CGMCC No.15196；
B2 the hybridoma of secondary antibody) is generated, deposit number is CGMCC No.15698；
C2 the hybridoma of third antibody) is generated, deposit number is CGMCC No.15699.
Hybridoma KZ-017-E6-2017-1226 is deposited in Chinese microorganism strain on December 27th, 2017 Preservation administration committee common micro-organisms center, depositary institution's address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation are compiled Number be CGMCC No.15196, the entitled hybridoma cell strain of the classification of the bacterial strain.
What hybridoma KZ-017-E6-2017-1226 was generated can specifically resist in conjunction with the first of spink1 Body includes light chain and heavy chain；
Light chain has the light chain CDR being made of 1CDR-L1,1CDR-L2,1CDR-L3；Heavy chain have by 1CDR-H1, The heavy chain CDR of 1CDR-H2,1CDR-H3 composition；
The amino acid sequence of 1CDR-L1,1CDR-L2,1CDR-L3 respectively as shown in SEQ ID NO.4,5 and 6, The amino acid sequence of 1CDR-H1,1CDR-H2,1CDR-H3 are respectively as shown in SEQ ID NO.7,8 and 9.Particular sequence information It is as shown in table 1 below:
The CDR information of 1 first antibody of table
Hybridoma KZN1-3C8-E8-2018-0507 was deposited in China Microbiological bacterium on May 08th, 2018 Kind preservation administration committee common micro-organisms center, depositary institution's address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation Number is CGMCC No.15698, the entitled hybridoma cell strain of the classification of the bacterial strain.
What hybridoma KZN1-3C8-E8-2018-0507 was generated can specifically combine the second of spink1 Antibody includes light chain and heavy chain；
Light chain has the light chain CDR being made of 2CDR-L1,2CDR-L2,2CDR-L3；Heavy chain have by 2CDR-H1, The heavy chain CDR of 2CDR-H2,2CDR-H3 composition；
The amino acid sequence of 2CDR-L1,2CDR-L2,2CDR-L3 respectively as shown in SEQ ID NO.10,11 and 12, The amino acid sequence of 2CDR-H1,2CDR-H2,2CDR-H3 are respectively as shown in SEQ ID NO.13,14 and 15.Particular sequence Information is as shown in table 2 below:
The CDR information of 2 secondary antibody of table
Sequence Sequence number
2CDR-L1 SSVSY SEQ ID NO.10
2CDR-L2 DTS SEQ ID NO.11
2CDR-L3 QQWRSNPPT SEQ ID NO.12
2CDR-H1 GFTFSSYG SEQ ID NO.13
2CDR-H2 INSNGGST SEQ ID NO.14
2CDR-H3 ARKITAWFAY SEQ ID NO.15
Hybridoma KZN3-5D7-D6-2018-0508 was deposited in China Microbiological bacterium on May 08th, 2018 Kind preservation administration committee common micro-organisms center, depositary institution's address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation Number is CGMCC No.15699, the entitled hybridoma cell strain of the classification of the bacterial strain.
The third that can specifically combine spink1 that hybridoma KZN3-5D7-D6-2018-0508 is generated Antibody includes light chain and heavy chain；
Light chain has the light chain CDR being made of 3CDR-L1,3CDR-L2,3CDR-L3；Heavy chain have by 3CDR-H1, The heavy chain CDR of 3CDR-H2,3CDR-H3 composition；
The amino acid sequence of 3CDR-L1,3CDR-L2,3CDR-L3 respectively as shown in SEQ ID NO.16,17 and 18, The amino acid sequence of 3CDR-H1,3CDR-H2,3CDR-H3 are respectively as shown in SEQ ID NO.19,20 and 21.Particular sequence Information is as shown in table 3 below:
The CDR information of 3 third antibody of table
Sequence Sequence number
3CDR-L1 QSVDYEGNSY SEQ ID NO.16
3CDR-L2 VAS SEQ ID NO.17
3CDR-L3 QQSNEDPWT SEQ ID NO.18
3CDR-H1 GYPFSRYW SEQ ID NO.19
3CDR-H2 IYPGDGDT SEQ ID NO.20
3CDR-H3 ARKGMLTPVDY SEQ ID NO.21
Hybridoma provided by the invention can generate antibody and its function fragment with above-mentioned CDR sequence, this is miscellaneous It hands over oncocyte that can stablize, efficiently produce the antibody and its function fragment of spink1 of high quality, can be used as engineered strain Carry out industrialized production.
A kind of isolated nucleic acid molecules are selected from following nucleic acid:
A3), DNA or RNA, encoding such antibodies and its function fragment；
B3 the nucleic acid of complementary nucleic acid defined in) and a3).
A kind of carrier comprising nucleic acid as described above.
The present invention further includes the core construct of at least one coding nucleic acid molecules as described above, preferred vector, into One step is preferably expression vector, such as plasmid.
A kind of host cell is converted by carrier as described above.In a preferred embodiment, host cell is Eukaryocyte, such as mammalian cell.
Above-mentioned spink1 antigen is in following a4) or b4) in application:
A4) preparation can specifically bind the antibody of spink1；
B4) screening is using spink1 albumen as the drug of target spot.
Above-mentioned antibody and its function fragment are in following a5)-d5) in application:
A5), for detecting the expression of spink1 albumen；
B5), as using spink1 albumen as the carrier of the drug of target spot；
C5), for purifying or separating spink1 albumen；
D5), it is used to prepare the product of detection spink1 albumen.
In certain embodiments of the present invention, the source of spink1 albumen be ox, it is horse, cow, pig, sheep, goat, big Mouse, mouse, dog, cat, rabbit, camel, donkey, deer, ermine, chicken, duck, goose, turkey, cockfighting or people.
In certain embodiments of the present invention, using above-mentioned antibody and its function fragment to the expression water of spink1 albumen The flat method detected includes western blot method, immunohistochemistry, immunostaining or ELISA method.
In certain embodiments of the present invention, using above-mentioned antibody and its function fragment to the expression water of spink1 albumen The flat sample detected includes whole blood, serum, blood plasma, urine or tumour vesica.
In certain embodiments of the present invention, preparation can be developed on the basis of above-mentioned antibody and its function fragment Spink1 albumen is the drug of target spot, and the drug is straight for treating liver cancer, oophoroma, cancer of pancreas, prostate cancer, breast cancer, knot Intestinal cancer, bladder cancer, gastric cancer or kidney etc..
In certain embodiments of the present invention, for identify, detect spink1 protein product include ELISA kit, Colloidal gold kit or paramagnetic particle method kit etc..
A kind of composition, including above-mentioned antibody and its function fragment and at least one diagnosticum and/or therapeutic agent, wherein Antibody and its function fragment and diagnosticum and/or therapeutic agent to form immune conjugate to be coupled.The composition is used for spink1 egg White identifies, purifies, detecting, separating or using spink1 as the research of target, drug etc..Due to the antibody and its functional sheet The high specific and potency, the composition of section can be applied to various fields and scene.
The present invention it is some be preferably carried out in mode, diagnosticum is selected from: radionuclide, radiocontrast medium, paramagnetic One of ion, metal, fluorescent marker, chemiluminescent labels, acoustic contrast agent or photosensitizer are a variety of.
In certain embodiments of the present invention, radionuclide includes110In、111In、177Lu、18F、52Fe、62Cu、64Cu、67Cu、67Ga、68Ga、86Y、90Y、89Zr、94mTc、94Tc、99mTc、120I、123I、124I、125I、131I、154-158Gd、32P、11C、13N、15O、186Re、188Re、51Mn、52mMn、55Co、72As、75Br、76Br、82MRb or83One of Sr or a variety of.
In certain embodiments of the present invention, paramagnetic ion includes chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or one of erbium (III) or a variety of.
In certain embodiments of the present invention, fluorescent marker include Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminacrine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5- carboxyl -4 ', 5 '-two chloro- 2 ', 7 ' - Dimethoxyfluorescein, 5- carboxyl -2 ', 4 ', 5 ', 7 '-tetrachlorofluoresceins, 5-carboxyfluorescein, 5- carboxyrhodamine, 6- carboxyrhodamine, 6- carboxyl tetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7,6-FAM, red sulphonyl Chlorine, fluorescein, HEX, 6-JOE, NBD (7- nitro benzo -2- oxa- -1,3- diazole), Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid (TPA), isophthalic diformazan Acid, cresols consolidate purple, cresols royal purple, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, It is succinylfluoresceins, rare earth metal cryptate, three pairs of pyridyl group diamines europiums, europium cryptate or chelate, diamines, double Anthocyanin, LaJolla indigo plant dyestuff, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamines, algae red Green white, phycoerythrin R, REG, rhodamine be green, rhodamine isothiocyanates, rhodamine are red, ROX, TAMRA, TET, TRIT (four Rhodamine isothiol), one of tetramethylrhodamine or texas Red or a variety of.
It is of the invention it is some be preferably carried out in mode, therapeutic agent is selected from: exposed antibody, cytotoxic agent, drug, radiation One of property nucleic, boron atom, immunomodulator, anti-apoptotic reagent, light sensitivity therapeutic agent, immune conjugate or oligonucleotides Or it is a variety of.
In certain embodiments of the present invention, drug is selected from methotrexate (MTX), fluorouracil, mercaptopurine, hydroxycarbamide, arabinose It is cytidine, mustargen, cyclophosphamide, thiotepa, cis-platinum, mitomycin, bleomycin, camptothecine, podophyllotoxin, actinomycin D, more It is soft than star, daunorubicin, vincaleukoblastinum, taxol, cephalotaxus alkaloid or l-Asparaginase.
In certain embodiments of the present invention, oligonucleotides is selected from one of shRNA, miRNA or siRNA or more Kind.
In certain embodiments of the present invention, immunomodulator is selected from: cell factor, chemotactic factor (CF), stem cell growth The factor, Hemopoietic factor, colony stimulating factor (CSF), interferon, hematopoietin, promotees thrombocytopoiesis at lymphotoxin Element, tumor necrosis factor (TNF), interleukin (IL), G-CSF (G-CSF), granular leukocyte macrophage- One of colony stimulating factor (GM-CSF) or stem cell factor are a variety of.
In certain embodiments of the present invention, radionuclide is selected from111In、111At、177Lu、211Bi、212Bi、213Bi 、211At、62Cu、67Cu、90Y、125I、131I、133I、32P、33P、47Sc、111Ag、67Ga、153Sm、161Tb、152Dy、166Dy、161Ho、166Ho、186Re、188Re、189Re、211Pb、212Pb、223Ra、225Ac、77As、89Sr、99Mo、105Rh、149Pm、169Er、194Ir、58Co 、80mBr、99mTc、103mRh、109Pt、119Sb、189mOs、192Ir、219Rn、215Po、221Fr、255Fm、11C、13N、15O、75Br、198Au、199Au、224Ac、77Br、113mIn、95Ru、97Ru、103Ru、105Ru、107Hg、203Hg、121mTe、122mTe、125mTe、165Tm 、167Tm、168Tm、197Pt、109Pd、142Pr、143Pr、161Tb、57Co、58Co、51Cr、59Fe、75Se、201Tl、76Br and169In Yb It is one or more.
Kit containing above-mentioned antibody and its function fragment can be, but not limited to as ELISA kit, colloid gold reagent Box or paramagnetic particle method kit, for detecting spink1 albumen.
In order to help to further understand the present invention, technical solution of the present invention is carried out now in conjunction with preferred embodiment detailed Explanation.
Unless otherwise specified, instrument or reagent commercially obtain in the embodiment of the present invention.The present invention is real It applies test method used in example unless otherwise specified, is conventional method.
Below by taking the spink1 antigen of SEQ ID NO.1 as an example, antigen selection process, first antibody and expression is described in detail The acquisition of its hybridoma, and antibody titer is detected.
1 Peptide systhesis of embodiment
Protein tertiary structure is carried out to spink1 albumen by I-TASSER protein tertiary structure software, as a result aa67- 79 regions are high hydrophilic region, it is contemplated that have strong antigen activity.The polypeptide chain KZ-017 of spink1 interception is synthesized (for spink1 Aa67-79, particular sequence are as follows: RQTSILIQKSGPC), purity: greater than 90%, wherein 5mg is coupled KLH, and about 5mg is used for for remaining ELISA.The polypeptide antigen and polypeptide for being coupled KLH are diluted to 1mg/ml with sterilizing PBS, and -20 DEG C save backup.Select peptide chain And non-recombinant protein more facilitates to lock antigenic determinant as antigen, mentions for subsequent ELISA pairing and antibody drug research For more theories integrations.
2 animal immune of embodiment
5 6-8 weeks male BALB/c mouses are immunized
1, initial immunity: KLH coupled peptide being mixed with isometric Freund's complete adjuvant, subcutaneous inoculation is carried out after emulsification, Every mouse subcutaneous injection 400ul emulsion.
2, be immunized for second: 1mg/mlKZ-017-KLH antigen is mixed with isometric freund 's incomplete adjuvant 1:1, cream Change, 200ul emulsion/mouse is injected intraperitoneally.
3, third time is immune: exempting from two.
4, vena ophthalmica takes blood, is placed at room temperature for 4000rpm after 2h, and 5min centrifugation takes serum.
5, coating KZ-017 polypeptide detects immune serum potency to 96 orifice plates, ELISA.
ELISA step:
1), polypeptide package amount is the hole 100ng/, and every hole 100ul, PBS dilution, 4 DEG C overnight.
2) it, outwells, adds confining liquid (10% skimmed milk power, PBS dissolution) hole 300ul/, 37 DEG C, 2h.
3), outwell, pat dry liquid, -20 DEG C freeze it is spare.
4) it, takes out the plank that has been coated in refrigerator to put to room temperature, the hole increase serum dilution 100ul/, 37 DEG C, 1h.
5), PBST (0.1%Tween) board-washing 3 times.
6), plus secondary antibody (horseradish enzyme marks goat anti-mouse) 1ul, 10mlPBS dilute, the hole 100ul/, and 37 DEG C, 45min.
7), PBST board-washing 3 times.
8), plus the hole TMB 100ul/, 37 DEG C, 15min or so.
9), plus the 2M hydrochloric acid hole terminate liquid 50ul/ terminates reaction.
10), 450nm wavelength surveys OD value.
ELISA the result shows that: KZ-017-KLH immune mice serum potency can be exempted from eventually.
6, exempt from eventually: selection 1#, 2# mouse, every intraperitoneal injection 200ugKZ-017-KLH antigen, 200ul antigen and 200ulPBS mixing.
3 cell fusion of embodiment
1, it recovers and cultivates murine myeloma cell (10%FBS+DMEM culture medium).
2, it takes and exempts from mouse spleen eventually, be prepared into individual cells suspension with culture medium, count.
3, by 1:5 mixing myeloma cell and mouse boosting cell, PEG is added to carry out cell fusion.
4, the hole 100ul/ 2 × HAT complete medium is spread into 96 orifice plates, 100ul fused cell liquid/hole is added.
5, it is measured in fused cell incubation with 1 × HAT half and changes liquid.
4 ELISA of embodiment screens cell hole supernatant
1, polypeptide package amount is the hole 100ng/, and every hole 100ul, PBS dilution, 4 DEG C overnight.It outwells, adding confining liquid, (10% is de- Rouge milk powder, PBS dissolution) hole 300ul/, 37 DEG C, 2h.
3, outwell, pat dry liquid, -20 DEG C freeze it is spare.
4, the plank that has been coated with is taken out in refrigerator to put to room temperature, add the hole cell supernatant 100ul/, 37 DEG C, 1h.
5, PBST (0.1%Tween) board-washing 3 times.
6, plus secondary antibody (horseradish enzyme marks goat anti-mouse) 1ul, 10mlPBS dilute, the hole 100ul/.37 DEG C, 45min.
7, PBST board-washing 3 times.
8, plus the hole TMB 100ul/, 37 DEG C, 15min or so.
9, plus the 2M hydrochloric acid hole terminate liquid 50ul/ terminates reaction.
10,450nm wavelength surveys OD value.
ELISA is as the result is shown: having multiple high positive clone holes.Select OD value high, clone is few, the good fusion hole of cell state 5 plants of cell, carry out the limiting dilution hole sizer selected monoclonal hybridoma of next step.
Embodiment 5 screens monoclonal hybridoma strain and hybridoma cell strain preservation
1, feeder cells are prepared from Balb/c or Kunming kind healthy mice (male and female).
2, the fusion hole cell that limiting dilution is chosen: with 1 × HT culture solution diluting cells, the cell suspension in every hole is added In 96 holes containing feeder cells, concentration is respectively the hole 100ul/ containing 0.75,1.5 and 3 cell, 5%CO2, 37 DEG C of cultures.
3, microscopically observation clonal growth situation after 10 days, ELISA detection have a cell clone hole, select positive value high and The hole of monoclonal (if necessary to do limiting dilution again, until all clone hole is positive).
4, culture expands selected monoclonal hybridoma and freeze-stored cell.
As a result: expanding and freeze KZ-017-E6-2017-1226.
5, the hybridoma is in preservation on December 27 in 2017 to China Committee for Culture Collection of Microorganisms's commonly micro- life Object center, preservation are registered on the books number are as follows: CGMCC No.15196.
The purifying of 6 antibody producing of embodiment
1, the pretreatment of BALB/c mouse: mouse is handled with the atoleine of sterilizing, 0.5mL/ is injected intraperitoneally only, 7-10 It is injectable hybridoma after it.
2, hybridoma is diluted to 1-2 × 10 with DMEM basic culture solution6A/milliliter, intraperitoneal injection of mice 0.5mL/ Only.
3, ascites acquires: observing mouse ascites production daily after 7 days and touches if abdomen obviously expands in interval When, skin has tension, can acquire ascites, primary every acquisition in one to two days.So repeatedly acquire repeatedly until mouse from It is so dead.Ascites centrifugation (2000rpm is centrifuged 5min), sucks the adipose tissue of top layer, removes cell component and others are heavy Starch.
4, ascites purifies: pretreated ascites is incubated for using Protein G pillar, elution.Finally obtain purifying Monoclonal antibody.
7 hybridoma CDR of embodiment sequencing
Source of mouse VH&VL is expanded from KZ-017-E6-2017-1226 cell, and target gene is connected into carrier T simultaneously Sequencing obtains first antibody CDR sequence:
Light Chain Antigen combined area:
1CDR-L1:ENIYSY (SEQ ID NO.4)
1CDR-L2:NAK (SEQ ID NO.5)
1CDR-L3:QHHYGIPWT (SEQ ID NO.6)
Heavy chain antigen binding domain:
1CDR-H1:GFTFSSFG (SEQ ID NO.7)
1CDR-H2:ISSGSSTL (SEQ ID NO.8)
1CDR-H3:DHGHRLL (SEQ ID NO.9)
8 antibody subtype of embodiment and titration
First antibody hypotype is IgG, kappa.
Antibody after purification is loaded two, SDS-PAGE glue and repeats purity testing, is analyzed through Image J, first Antibody purity is greater than 95%.As a result as shown in Figure 1.
It is 0.6mg/ml, the minimum effect of potency 1:80000, i.e. first antibody through NanoDrop measurement first antibody concentration Concentration is 0.0075ug/ml, better than antibody (minimum activity 0.8ug/ml) in the market.The titration result of antibody is as follows Shown in table:
It should be understood that positive control is fusion serum, dilution 1:1000, negative control is healthy mice blood Clear 1:1000 dilution.
The spink1 antigen of 9 SEQ ID NO.2 of embodiment
Based on identical method in embodiment 1, the polypeptide chain KZN1 of spink1 interception is synthesized (for spink1aa1-18, tool Body sequence are as follows: MKVTGIFLLSALALLSLS), select the reason of this section of peptide chain is as antigen production antibody are as follows: research shows that liver The expression molecular weight (about 10kd) of spink1 is greater than expected molecular weight (about 6.3kd) in cancer cell, and sequencing result shows the molecule Amount variation may be since N-terminal signal peptide peptide chain is not caused by complete excision in liver cancer cells.Therefore this section of sequence is chosen to increase Identify the specificity of liver cancer cells.
KZN1 is tested referring to the experimental procedure of embodiment 2-5, as a result successfully constructs hybridoma KZN1- 3C8-E8-2018-0507, the hybridoma are entrusted in the preservation of May 8 in 2018 to Chinese microorganism strain preservation management Member can common micro-organisms center, preservation registers on the books number are as follows: CGMCC No.15698.
Experimental procedure referring to embodiment 6-7 carries out purifying to secondary antibody and CDR is sequenced:
Light Chain Antigen combined area:
2CDR-L1:SSVSY (SEQ ID NO.10)
2CDR-L2:DTS (SEQ ID NO.11)
2CDR-L3:QQWRSNPPT (SEQ ID NO.12)
Heavy chain antigen binding domain:
2CDR-H1:GFTFSSYG (SEQ ID NO.13)
2CDR-H2:INSNGGST (SEQ ID NO.14)
2CDR-H3:ARKITAWFAY (SEQ ID NO.15)
Secondary antibody hypotype is IgM, kappa.
Secondary antibody after purification is loaded two, SDS-PAGE glue and repeats purity testing, is analyzed through ImageJ pure Degree is greater than 90%.As a result as shown in Figure 2.
It is 0.18mg/ml, the minimum effect of potency 1:4000, i.e. secondary antibody through NanoDrop measurement secondary antibody concentration Concentration is 0.045ug/ml.The titration result of antibody is as shown in the table:
KZN1-3C8-E8-2018-0507
1:500 1.095
1:1000 0.878
1:2000 0.528
1:4000 0.267
1:8000 0.197
1:16000 0.178
PBS 0.183
The spink1 antigen of 10 SEQ ID NO.3 of embodiment
Based on identical method in embodiment 1, synthesize spink1 interception polypeptide chain KZN3 (for spink1aa19-35, Particular sequence are as follows: GNTGADSLGREAKCYNE), select the reason of this section of peptide chain is as antigen production antibody are as follows: this section of peptide chain is more Easily sudden and violent leakage has a strong antigen on the outside of spink1 albumen, at the same the N-terminal peptide chain determined be antibody medicine research and develop provide more information and It is easier to be formed with C-terminal antibody in ELAISA double-antibody method complementary.
KZN1 is tested referring to the experimental procedure of embodiment 2-5, as a result successfully constructs hybridoma KZN3- 5D7-D6-2018-0508, the hybridoma are entrusted in the preservation of May 8 in 2018 to Chinese microorganism strain preservation management Member can common micro-organisms center, preservation registers on the books number are as follows: CGMCC No.15699.
Experimental procedure referring to embodiment 6-7 carries out purifying to secondary antibody and CDR is sequenced:
Light Chain Antigen combined area:
3CDR-L1:QSVDYEGNSY (SEQ ID NO.16)
3CDR-L2:VAS (SEQ ID NO.17)
3CDR-L3:QQSNEDPWT (SEQ ID NO.18)
Heavy chain antigen binding domain:
3CDR-H1:GYPFSRYW (SEQ ID NO.19)
3CDR-H2:IYPGDGDT (SEQ ID NO.20)
3CDR-H3:ARKGMLTPVDY (SEQ ID NO.21)
Third antibody subtype is IgG1, kappa.
Third antibody after purification is loaded two, SDS-PAGE glue and repeats purity testing, is analyzed through Image J pure Degree is greater than 90%.As a result as shown in Figure 3.
It is 0.3mg/ml, potency 1:640000, the i.e. minimum work of third antibody through NanoDrop measurement third antibody concentration It is 0.00046875ug/ml with concentration.The titration result of antibody is as shown in the table:
KZN3-5D7-D6-2018-0508 KZN3-5D7-D6-2018-0508
1:10000 3.654 3.575
1:20000 3.637 3.546
1:40000 3.595 3.557
1:80000 3.559 3.516
1:160000 3.485 3.486
1:320000 3.168 2.982
1:640000 2.205 2.08
PBS 0.178 0.168
The western blot of first antibody in 11 KZ-017-E6-2017-1226 cell of embodiment
1.SDS-PAGE
5% concentration+15% separation gel of glue (containing 10% glycerol), 80V 100min, 110V 60min.10 μ L of loading volume.
Sample treatment: liver cancer patient blood sample, normal blood sample, to take supernatant after isometric acetonitrile precipitation high molecular weight protein, 100 It DEG C uncaps and to boil sample 3-5min.
0 DEG C of Spink1 protein 10 is boiled sample 3-5min.4 μ g of applied sample amount.
2. transferring film
100V is wet to turn 40min.
3. closing
5% skim milk (PBST) room temperature shaker closes 2h.
4. primary antibody is incubated for
It is 1 μ g/mL with 5% skim milk (PBST) dilution primary antibody, 4 DEG C overnight.
5. washing is three times, 5min is each.
6. secondary antibody is incubated for
It is 1:5000, room temperature shaker 1h with PBST dilution sheep anti mouse-HRP.
7. washing is three times, 5min is each.
8. exposure
ECL exposure, observes band, as a result as shown in Figure 4.Test result shows that first antibody can identify liver cancer simultaneously Cell and the spink1 albumen of plant cell expression.
The western blot of third antibody in 12 KZN3-5D7-D6-2018-0508 cell of embodiment
1.SDS-PAGE
5% concentration+15% separation gel of glue (containing 10% glycerol), 80V 100min, 110V 60min.10 μ L of loading volume.
Sample treatment: liver cancer patient blood sample, normal blood sample, to take supernatant after isometric acetonitrile precipitation high molecular weight protein, 100 It DEG C uncaps and to boil sample 3-5min.
0 DEG C of Spink1 protein 10 is boiled sample 3-5min.4 μ g of applied sample amount.
2. transferring film
100V is wet to turn 40min.
3. closing
5% skim milk (PBST) room temperature shaker closes 2h.
4. primary antibody is incubated for
It is 1 μ g/mL with 5% skim milk (PBST) dilution primary antibody, 4 DEG C overnight.
5. washing is three times, 5min is each.
6. secondary antibody is incubated for
It is 1:5000, room temperature shaker 1h with PBST dilution sheep anti mouse-HRP.
7. washing is three times, 5min is each.
8. exposure
ECL exposure, observes band, as a result as shown in figure 5, test result shows that kzn3 antibody can identify liver cancer cells The spink1 albumen of expression.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Hao Ling Saiao (Tianjin) Biotechnology Co., Ltd
Tianjin edge is to Biotechnology Co., Ltd
<120>spink1 antigen, antibody and its function fragment and its application and product that spink1 can be specifically bound
<160> 21
<170> PatentIn version 3.5
<210> 1
<211> 13
<212> PRT
<213>artificial sequence
<400> 1
Arg Gln Thr Ser Ile Leu Ile Gln Lys Ser Gly Pro Cys
1 5 10
<210> 2
<211> 18
<212> PRT
<213>artificial sequence
<400> 2
Met Lys Val Thr Gly Ile Phe Leu Leu Ser Ala Leu Ala Leu Leu Ser
1 5 10 15
Leu Ser
<210> 3
<211> 17
<212> PRT
<213>artificial sequence
<400> 3
Gly Asn Thr Gly Ala Asp Ser Leu Gly Arg Glu Ala Lys Cys Tyr Asn
1 5 10 15
Glu
<210> 4
<211> 6
<212> PRT
<213>artificial sequence
<400> 4
Glu Asn Ile Tyr Ser Tyr
1 5
<210> 5
<211> 3
<212> PRT
<213>artificial sequence
<400> 5
Asn Ala Lys
1
<210> 6
<211> 9
<212> PRT
<213>artificial sequence
<400> 6
Gln His His Tyr Gly Ile Pro Trp Thr
1 5
<210> 7
<211> 8
<212> PRT
<213>artificial sequence
<400> 7
Gly Phe Thr Phe Ser Ser Phe Gly
1 5
<210> 8
<211> 8
<212> PRT
<213>artificial sequence
<400> 8
Ile Ser Ser Gly Ser Ser Thr Leu
1 5
<210> 9
<211> 7
<212> PRT
<213>artificial sequence
<400> 9
Asp His Gly His Arg Leu Leu
1 5
<210> 10
<211> 5
<212> PRT
<213>artificial sequence
<400> 10
Ser Ser Val Ser Tyr
1 5
<210> 11
<211> 3
<212> PRT
<213>artificial sequence
<400> 11
Asp Thr Ser
1
<210> 12
<211> 9
<212> PRT
<213>artificial sequence
<400> 12
Gln Gln Trp Arg Ser Asn Pro Pro Thr
1 5
<210> 13
<211> 8
<212> PRT
<213>artificial sequence
<400> 13
Gly Phe Thr Phe Ser Ser Tyr Gly
1 5
<210> 14
<211> 8
<212> PRT
<213>artificial sequence
<400> 14
Ile Asn Ser Asn Gly Gly Ser Thr
1 5
<210> 15
<211> 10
<212> PRT
<213>artificial sequence
<400> 15
Ala Arg Lys Ile Thr Ala Trp Phe Ala Tyr
1 5 10
<210> 16
<211> 10
<212> PRT
<213>artificial sequence
<400> 16
Gln Ser Val Asp Tyr Glu Gly Asn Ser Tyr
1 5 10
<210> 17
<211> 3
<212> PRT
<213>artificial sequence
<400> 17
Val Ala Ser
1
<210> 18
<211> 9
<212> PRT
<213>artificial sequence
<400> 18
Gln Gln Ser Asn Glu Asp Pro Trp Thr
1 5
<210> 19
<211> 8
<212> PRT
<213>artificial sequence
<400> 19
Gly Tyr Pro Phe Ser Arg Tyr Trp
1 5
<210> 20
<211> 8
<212> PRT
<213>artificial sequence
<400> 20
Ile Tyr Pro Gly Asp Gly Asp Thr
1 5
<210> 21
<211> 11
<212> PRT
<213>artificial sequence
<400> 21
Ala Arg Lys Gly Met Leu Thr Pro Val Asp Tyr
1 5 10

Claims (10)

1. a kind of spink1 antigen, which is characterized in that be following a)-f) in any polypeptide:
a)RQTSILIQKSGPC(SEQ ID NO.1)；
B) by amino acid sequence shown in SEQ ID NO.1 by one or several amino acid residues substitution and/or missing and/ Or add and have the polypeptide as derived from a) of spink1 epitope function；
c)MKVTGIFLLSALALLSLS(SEQ ID NO.2)；
D) by amino acid sequence shown in SEQ ID NO.2 by one or several amino acid residues substitution and/or missing and/ Or add and have the polypeptide as derived from c) of spink1 epitope function；
e)GNTGADSLGREAKCYNE(SEQ ID NO.3)；
F) by amino acid sequence shown in SEQ ID NO.3 by one or several amino acid residues substitution and/or missing and/ Or add and have the polypeptide as derived from e) of spink1 epitope function.
2. antibody and its function fragment that one kind can specifically bind spink1, which is characterized in that be following a1)-c1) in Any antibody and its function fragment:
A1) the first antibody of SEQ ID NO.1, the first antibody and its function fragment include light chain and heavy chain；
The light chain has the light chain CDR being made of 1CDR-L1,1CDR-L2,1CDR-L3；The heavy chain have by The heavy chain CDR of 1CDR-H1,1CDR-H2,1CDR-H3 composition；
The amino acid sequence of described 1CDR-L1,1CDR-L2,1CDR-L3 are described respectively as shown in SEQ ID NO.4,5 and 6 The amino acid sequence of 1CDR-H1,1CDR-H2,1CDR-H3 are respectively as shown in SEQ ID NO.7,8 and 9；
B1) the secondary antibody of SEQ ID NO.2, the secondary antibody and its function fragment include light chain and heavy chain；
The light chain has the light chain CDR being made of 2CDR-L1,2CDR-L2,2CDR-L3；The heavy chain have by The heavy chain CDR of 2CDR-H1,2CDR-H2,2CDR-H3 composition；
The amino acid sequence of described 2CDR-L1,2CDR-L2,2CDR-L3 respectively as shown in SEQ ID NO.10,11 and 12, The amino acid sequence of described 2CDR-H1,2CDR-H2,2CDR-H3 are respectively as shown in SEQ ID NO.13,14 and 15；
C1) the third antibody of SEQ ID NO.2, the third antibody and its function fragment include light chain and heavy chain；
The light chain has the light chain CDR being made of 3CDR-L1,3CDR-L2,3CDR-L3；The heavy chain have by The heavy chain CDR of 3CDR-H1,3CDR-H2,3CDR-H3 composition；
The amino acid sequence of described 3CDR-L1,3CDR-L2,3CDR-L3 respectively as shown in SEQ ID NO.16,17 and 18, The amino acid sequence of described 3CDR-H1,3CDR-H2,3CDR-H3 are respectively as shown in SEQ ID NO.19,20 and 21.
3. antibody according to claim 2 and its function fragment, which is characterized in that the antibody and its function fragment include Spink1 chimeric antibody and its function fragment；
Preferably, the antibody include ox, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, Ermine, chicken, duck, goose, turkey, cockfighting or people IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD any one of them is permanent Determine the sequence in area；
Preferably, the function fragment includes F (ab ')2, Fab ', Fab, Fv, scFv, bispecific antibody and the identification of antibody minimum it is single One of position is a variety of.
4. a kind of hybridization for generating the antibody and its function fragment described in claim 2 or 3 that can specifically bind spink1 Oncocyte, which is characterized in that be following a2)-c2) in any hybridoma:
A2 the hybridoma of first antibody, deposit number CGMCCNo.15196) are generated；
B2 the hybridoma of secondary antibody, deposit number CGMCCNo.15698) are generated；
C2 the hybridoma of third antibody, deposit number CGMCCNo.15699) are generated.
5. a kind of isolated nucleic acid molecules, which is characterized in that the nucleic acid molecules are selected from following nucleic acid:
A3), DNA or RNA encodes antibody and its function fragment described in claim 2 or 3；
B3 the nucleic acid of complementary nucleic acid defined in) and a3).
6. a kind of carrier, which is characterized in that including the nucleic acid molecules described in claim 5.
7. spink1 antigen described in claim 1 is in following a4) or b4) in application:
A4) preparation can specifically bind the antibody of spink1；
B4) screening is using spink1 albumen as the drug of target spot.
8. antibody and its function fragment described in Claims 2 or 3 are in following a5)-d5) in application:
A5), for detecting the expression of spink1 albumen；
B5), as using spink1 albumen as the carrier of the drug of target spot；
C5), for purifying or separating spink1 albumen；
D5), it is used to prepare the product of detection spink1 albumen；
Preferably, the source of the spink1 albumen be ox, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, Camel, donkey, deer, ermine, chicken, duck, goose, turkey, cockfighting or people；
Preferably, the a5) in the method for detection include westernblot method, immunohistochemistry, immunostaining or ELISA method；
Preferably, the a5) in the sample of detection include whole blood, serum, blood plasma, urine or tumour vesica；
Preferably, the b5) in drug for treating liver cancer, oophoroma, cancer of pancreas, prostate cancer, breast cancer, Colon and rectum Cancer, bladder cancer, gastric cancer or kidney；
Preferably, the d5) in product include ELISA kit, colloidal gold kit or paramagnetic particle method kit.
9. a kind of composition, which is characterized in that the composition include antibody and its function fragment described in Claims 2 or 3 and At least one diagnosticum and/or therapeutic agent；
The antibody and its function fragment and diagnosticum and/or therapeutic agent to form immune conjugate to be coupled.
10. a kind of kit containing antibody described in Claims 2 or 3 and its function fragment, which is characterized in that the kit For ELISA kit, colloidal gold kit or paramagnetic particle method kit, the kit is for detecting spink1 albumen.
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WO2021007338A1 (en) * 2019-07-08 2021-01-14 Imcare Biotech, Llc. Anti-serine protease inhibitor kazal (spik) antibodies, immunoconjugates, and methods of use
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