CN112251444B

CN112251444B - Modified AMH gene sequence and method for preparing AMH by using same

Info

Publication number: CN112251444B
Application number: CN202011129577.4A
Authority: CN
Inventors: 徐万熙; 冉晓园; 程若东; 王玉满; 王刚
Original assignee: Kaikai Biotechnology Shanghai Co ltd
Current assignee: Kaikai Biotechnology Shanghai Co ltd
Priority date: 2020-10-21
Filing date: 2020-10-21
Publication date: 2023-05-16
Anticipated expiration: 2040-10-21
Also published as: CN112251444A

Abstract

The invention provides an improved AMH gene sequence and an amino acid sequence, which respectively have a sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2, and a sequence shown as SEQ ID NO. 3 or SEQ ID NO. 4. The invention also discloses a method for obtaining AMH by coexpression of AMH gene sequence and receptor protein AMHRII _N,C Pro-AMH protein. According to the invention, AMH protein can be prepared at a high level by optimizing high-expression exogenous signal peptide, designing sequences near furin tangent points and adopting an AMHRII receptor coexpression strategy.

Description

Modified AMH gene sequence and method for preparing AMH by using same

Technical Field

The invention belongs to the technical field of bioengineering, in particular to an improved AMH gene sequence and amino acid, and the AMH obtained by coexpression of the modified AMH gene sequence and amino acid and receptor protein AMHRII _N,C Pro-AMH protein.

Background

Anti Lesion tube hormone (AMH), also known as Lesion tube inhibitor (MIS), is a transforming growth factor beta (TGF-. Beta.) superfamily member, and AMH is synthesized mainly in granulosa cells of primary and pre-sinus follicles, closely related to ovarian function and stores. It is not regulated by hypothalamus-pituitary-ovary axis, has no periodic variation in menstrual cycle and constant level, and compared with other traditional biological indexes, AMH is the most accurate biomarker for evaluating the quality of ovaries.

AMH is intermolecular disulfide bond connected dimer, has molecular weight of 140kDa, and is prepared by treating furin fu in human bodyrin (PCSK 3) or PCSK5/PC5 cleavage, AMH is cleaved into 110kDa N-terminal dimer protein (AMH) _N ) And 25kDa C-terminal dimer protein (AMH _C )。

AMH _N And AMH _C Protein complexes (AMH) formed by non-covalent bonding _N,C ) Is a functional active protein. The human body also has an uncleaved covalently linked full-length protein (Pro-AMH), which is a low-activity protein. The natural protein is purified and extracted, the yield is extremely low, the cost is high, and the two different forms of AMH proteins with different functions cannot be separated in a distinguishing way (US 005310880A). Recombinant expression of AMH _N,C The protein expression level is low and difficult to obtain. Recombinant expression of AMH has been reported in literature and patents _N,C The main strategy (US 005359033A; PNAS, vol.93, pp.7711-7716,1996) is to promote cleavage efficiency by optimizing the furin (furin) cleavage site sequence of AMH. Through co-expression with PC5, the digestion efficiency is promoted; AMH is improved by establishing and screening CHO stable transgenic cell lines _N,C Protein expression level. However, purification preparation yields mg-grade recombinant AMH _N,C Proteins remain very difficult and do not meet the requirements of antibody development and IVD detection applications.

Therefore, there is an urgent need in the art to solve the problem of increasing the expression level of AMH.

Disclosure of Invention

In order to solve the defects in the prior art, the invention obviously improves the expression quantity of AMH by coexpression of AMH and receptor protein AMHRII. AMH can be obtained by co-expression of AMH sequence 1 and AMHRII by designing the sequence near the tangent point of AMH furin _N,C The method comprises the steps of carrying out a first treatment on the surface of the Co-expression of AMH sequence 2 with AMHRII can result in Pro-AMH. Transient transfection of shake flask expression with mammalian Expi293 allows purification to achieve mg-grade AMH _N,C Pro-AMH protein. Specifically, the invention provides the following technical scheme:

in one aspect, the invention provides an engineered AMH gene sequence characterized in that: the sequence of the modified AMH gene is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

Preferably, the AMH gene sequence is devoid of a signal peptide sequence.

Preferably, the signal peptide sequence of SEQ ID NO. 1 or SEQ ID NO. 2 may be replaced with any suitable signal peptide sequence, preferably a high expression exogenous signal peptide. The selection of suitable signal peptides according to the different expression conditions is within the ability of the person skilled in the art and is also within the scope of the present invention.

In another aspect, the invention provides an engineered AMH amino acid sequence characterized in that: the sequence of the modified AMH gene is shown as SEQ ID NO. 3 or SEQ ID NO. 4.

Preferably, the AMH amino acid sequence is devoid of a signal peptide sequence.

Preferably, the signal peptide sequence of SEQ ID NO. 3 or SEQ ID NO. 4 may be replaced with any suitable signal peptide sequence, preferably a high expression exogenous signal peptide. The selection of suitable signal peptides according to the different expression conditions is within the ability of the person skilled in the art and is also within the scope of the present invention.

In yet another aspect, the invention provides an expression vector characterized by: the expression vector comprises the AMH gene sequence of claim 1.

Preferably, the expression vector is a mammalian cell expression vector.

Preferably, mammalian cell expression vectors may be selected from pCDNA3.1, pCMV, and any other expression vector. Those skilled in the art will be able to select any suitable expression vector according to the circumstances without departing from the scope of the invention.

In a further aspect, the invention provides the use of an AMH gene sequence according to the invention for the preparation of AMH. The use prepares AMH, for example by means of co-expression.

In yet another aspect, the present invention provides a method of preparing AMH, characterized by: coexpression of the AMH gene sequence of claim 1 with the receptor protein AMHRII gene sequence.

Preferably: the method comprises the following steps:

s1, constructing the AMH gene sequence and the receptor protein AMHRII gene sequence of claim 1 into a mammalian cell expression vector;

s2, transiently transfecting the mammalian cell expression vector in the step S1 into mammalian cells;

s3 expression of AMH _N,C Pro-AMH protein.

Preferably, the AMH gene sequence and the receptor protein AMHRII gene sequence are set forth in 4: 1. 3: 1. 2: 1. 1: 1. 1: 2. 1: 3. 1: 4.

More preferably, the AMH gene sequence and the receptor protein AMHRII gene sequence are set forth in 2:1, co-transfection.

It will be appreciated that the present invention may be co-transfected in any suitable ratio without departing from the scope of the invention.

Preferably, the mammalian cell is selected from an Expi293 cell or a CHO cell. It should be understood that any suitable mammalian cell may be employed without departing from the scope of the invention.

The invention has the remarkable technical effects that:

the method adopts AMHRII and AMH sequence 1 or AMHRII and AMH sequence 2 to co-express, and the method transiently transfects an Expi293 cell, and cell secretion supernatant is detected to be positive by anti-His tag WB. Amplified cell transfection in shake flask, and can be purified to obtain AMH _N,C And ProAMH. Under the same transfection expression condition, the Wild Type (WT) AMH sequence is independently transfected and expressed, or the furin tangent point in the expression literature is optimized, or the AMH sequence 1, the AMH sequence 2 and the WB are both negative; all the sequences are respectively and transiently transfected with furin to be expressed by the reference and the prior patent method, and the detection of the cell secretion supernatant WB is negative.

The literature methods are all to stably transfer cell lines to express and prepare AMH, and mammalian cells are transiently transfected according to literature related sequences, and WB detection is negative. The method disclosed by the invention is used for carrying out design optimization on furin tangent points and sequences near furin tangent points in the AMH sequence 1 and the AMH sequence 2, exposing the furin tangent points, and effectively cutting the AMH sequence 1 by using endogenous furin protein of an Expi293 cell.

The method adopts AMHRII<Fc>The fusion protein and the AMH sequence 1 and the AMH sequence 2 fused with the N-end His tag are respectively co-expressed. AMHRII<Fc>Or AMHRII<Fc>the/AMHc complex protein can be separated and purified by protein A resin affinity. Isolation and enrichment of AMHRII<Fc>Or AMH (automated mechanical transmission)RII<Fc>After the/AMHc complex, the cell supernatant is purified and enriched with AMH by Ni chelating resin _N,C Or ProAMH, and purifying with further ion column or hydrophobic column to obtain recombinant protein AMH _N,C Or ProAMH.

Drawings

FIG. 1 is an AMH _N,C And ProAMH mammalian cell Expi293 transient transfection and protein expression detection results.

anti-His tagged WB detection of the secretion supernatant from the Expi293 mammalian cells, it could be detected<His>AMH _N,C And<His>secretory expression of ProAMH,

lane 1:<His>AMH _N,C reducing electrophoresis,

lane 2: < His > ProAMH, reduction electrophoresis.

FIG. 2 shows the results of examination of AMH sequence 1 and receptor AMHRII-Fc co-expression.

AMH sequence 1 and receptor AMHRII-Fc are co-expressed, cell expression supernatant is purified by protein A affinity column, and AMHRII-Fc protein and AMHRII-Fc-AMHc complex are enriched and hung. Indicating AMH _N,C Expression was obtained and AMHRII-Fc competed for binding to a certain amount of AMHc. As shown in lanes a and B of fig. 2.

The protein A affinity column fluid<His tag>AMH _N,C Purifying with Ni chelating affinity column, and purifying with ion column to obtain recombinant protein<His tag>AMH _N,C . As shown in lanes C and D of fig. 2.

Protein A affinity column purification lanes A, B,

lane a: AMHRII-Fc-AMHc complex, reduction electrophoresis,

lane B: AMHRII-Fc-AMHc complex, non-reducing electrophoresis.

Arrow 5 indicates the AMHRII-Fc protein in SDS-page lane A, reduction electrophoresis,

arrow 6 indicates the AMHRII-Fc protein in SDS-page lane B, non-reducing electrophoresis.

The AMHc protein indicated by arrow 1 can be observed in lane a, electrophoresis by reduction,

the AMHc protein indicated by arrow 2, non-reducing electrophoresis, can be observed in lane B.

Lane A arrow 5 indicates AMHRII-Fc over-expression, binding to a certain amount of AMHc protein (arrow 1),

lane B arrow 6 indicates dimer formation of AMHRII-Fc non-reduced sample; arrow 2 indicates dimer formation in AMHc non-reduced samples.

Ni chelate affinity column purification lane C, lane D,

lane C:<His>AMH _N,C the complex, the reduction electrophoresis,

lane D:<His>AMH _N,C complex, non-reducing electrophoresis.

AMH indicated by arrow 3 can be observed in lane C _N Protein, AMHC protein indicated by arrow 1, reduction electrophoresis,

AMH indicated by arrow 4 can be observed in lane D _N Dimeric protein, AMH indicated by arrow 2 _C Dimer protein, non-reducing electrophoresis.

FIG. 3 shows the results of examination of AMH sequence 2 and receptor AMHRII-Fc co-expression.

AMH sequence 2 and receptor AMHRII-Fc are expressed together, cell expression supernatant is purified by protein A affinity column, and AMHRII-Fc protein is enriched and hung column is obtained. Protein A is purified and flowed through an affinity column, then is purified by a Ni chelating affinity column, and Ni column eluted Protein is further purified by an ion column to obtain recombinant Pro-AMH Protein (containing His tag affinity tag) which is shown in SDS-page (figure 3) below, the apparent molecular weight of the reduced electrophoresis sample Protein is 70kDa, the non-reduced electrophoresis sample Protein is in the form of disulfide bond connected dimer, and the apparent molecular weight is 140kDa.

Detailed Description

The invention will be further illustrated by the following specific examples, which are to be understood as illustrative only and are not limiting of the invention.

The starting materials and equipment used in the examples are well known to those skilled in the art and are commercially available or readily available or available.

Experimental materials:

Expi293 ^TM expression System kit high density cell mammalian expressionThe system comprises: thermo Fisher Scientific

Protein a resin: GE healthcare

Nickel-loaded IMAC resin: GE healthcare

Phenyl-HP pre-packed column: GE healthcare

Protein A280 concentration determination Nanodrop Thermo Fisher Scientific

Imidazole (imidozole): sigma-Aldrich

Phosphate, tris-base, naCl and other chemical reagents: group of Chinese medicine

SDS-page gel electrophoresis system: tanon Shanghai energy technology Co.Ltd

anti-His-tag antibody: jinsri biotechnology Co Ltd

Millipore 3kDa cutoff concentration tube: merck.

EXAMPLE 1 Gene synthesis and construction of mammalian expression vectors

The AMH sequence 1 and AMH sequence 2 genes were synthesized by general biosystems (anhui) limited. The start codon ATG was preceded by a Kozac sequence and constructed into the pTT5 mammalian expression vector at EcoRI/HindIII sites. Sequencing was correct.

The exogenous signal peptide is not limited to the preferred signal peptide disclosed in the patent, and can be screened out by mammalian cell expression detection, so that the signal peptide with good secretion and expression effects on the AMH sequence 1 and the AMH sequence 2 proteins can be screened out.

Other expression vectors such as pCDNA3.1 and pCMV can be selected as the mammalian expression vector.

Example 2 mammalian Expi293 transient transfection and protein expression detection

Expi293 cell cultures and transfection employed the experimental conditions recommended in the product manual of the Expi293 expression system (Ref: MAN 0007814). The cells were fed on day 2 after transfection, harvested on day 6 and centrifuged at 10000rpm to obtain the cell supernatant.

AMH sequence 1 expression plasmid and AMH RII-Fc expression plasmid were combined in 2:1, and plasmid ratio can be adjusted to promote expression of AMH sequence 1.

AMH sequence 2 expression plasmid and AMH RII-Fc expression plasmid were expressed as 2:1, and plasmid ratio can be adjusted to promote expression of AMH sequence 2.

The cell expression supernatants from the pilot expression (i.e., transient transfection of 1-5ml cells) were enriched with one-step nickel-charged IMAC resin, eluted, and the anti-His tag WB results are shown in FIG. 1. To judge AMH _N,C And Pro-AMH expression. After successful expression of the protein of interest is determined, the cells are transfected with the amplification and 1-10L of cells are transfected. And (3) purifying by adopting a purification method in the step 3 to obtain the protein.

Sequence design near furin tangent point of AMH sequence 1, FIG. 1 shows that protein expression WB assay lane HLQSSRHRRALD (FIG. 1) shows that endogenous furin protein from an Expi293 cell is sufficient to cleave AMH sequence 1 to form AMH _N,C WB detection can detect<His>AMH _N Strip, AMH _N And AMH (advanced mechanical systems) _C Has been completely cut. WB did not detect the non-cleaved AMH sequence 1 protein.

The furin tangent point sequence RAQR in the furin tangent point sequence HLQSGGGGGALD, WT AMH sequence in AMH sequence 2 is replaced by GGGG four amino acids, and protein expression WB detection lane 2 (FIG. 1) shows that ProAMH obtains secretory expression.

The AMH sequence 1 and the AMH sequence 2 of the patent can be used for establishing CHO stable transgenic expression strains, and further improving the protein expression quantity.

EXAMPLE 3 AMH _N,C Pro-AMH protein purification

1. AMH (advanced mechanical systems) _N,C Protein purification

a. The shake flask was transiently transfected with the Expi293 cells, 1L of secretion supernatant was harvested, pre-packed with 5ml of protein A in column volume, hung in column, equilibrated buffer 20mM PB,500mM NaCl,pH7.4, 5 column volumes were washed with equilibration buffer after secretion supernatant was loaded, and protein enriched on protein A column was eluted with 0.1M glycine, pH2.9 buffer and neutralized with 1M Tris-Base buffer. Dialyzing the eluted protein to PBS, buffer solution with pH7.4, concentrating with 3kDa cutoff concentrating tube to obtain AMH RII-Fc/AMH _C Complex proteins. As shown in fig. 2 lanes a, B.

b. In the previous purification step a, the column of the ProteinA column was run through a nickel-loaded IMAC pre-packed column with a column volume of 5ml, the column was hung, equilibration buffer 20mM PB,500mM NaCl, pH7.4, 5 column volumes with 20mM PB,500mM NaCl,20mM imidazole, pH7.4 buffer, 5 column volumes with 20mM PB,500mM NaCl,50mM imidazole, pH7.4 buffer. Elution was performed with 20mM PB,500mM NaCl,500mM imidazole, pH7.4 buffer. Dialyzing the eluted protein to PBS, buffer solution with pH7.4, concentrating with 3kDa cutoff concentrating tube to obtain AMH _N,C Complex proteins. As shown in fig. 2 lanes C, D.

AMH _N,C The purification yield of the complex protein is 0.5mg/L.

2. Pro-AMH protein purification

a. The secretion supernatant was harvested by shake flask transient transfection of the Expi293 cells, 1L of the secretion supernatant was hung on a nickel-loaded IMAC pre-packed column with a column volume of 5ml, equilibration buffer 20mM PB,500mM NaCl,pH7.4, 5 column volumes were washed with 20mM PB,500mM NaCl,20mM imidazole, pH7.4 buffer, 5 column volumes were washed with 20mM PB,500mM NaCl,50mM imidazole, pH7.4 buffer, 20mM PB,500mM NaCl,500mM imidazole, pH7.4 buffer, respectively, and eluted.

b. The previous purification step a. Elution of the protein (6-10 ml) was dialyzed into 20mM PB,3M NaCl,pH7.4. Column-mounted Phenyl-HP with a column volume of 1ml, gradient elution with 20mM PB, pH7.4 buffer. The target protein Pro-AMH was eluted at a salt concentration gradient of 20mM PB, pH7.4 buffer 1M-500 mM. The eluted protein was dialyzed against PBS, pH7.4 buffer, and concentrated in a 3kDa cutoff tube to obtain ProAMH complex protein. As shown in fig. 3, lanes N, R.

The Pro-AMH protein purification yield was 0.2mg/L.

The AMH protein preparation method of the invention can stably obtain AMH _N,C And a purification yield of ProAMH protein of 0.2-0.5 mg/L.

Preferably, the high-expression exogenous signal peptide, the sequence design near furin tangent point and the AMHRII receptor co-expression strategy are three key factors for obtaining AMH protein, and are innovation points of the invention.

AMH sequence 1 (SEQ ID NO: 3)

Single underline is the exogenous signal peptide; the double underlined sequence is specially designed by the applicant, and RHRR is furin tangent point selected by the applicant. Italics indicates the design of the sequence around the furin tangent point.

AMH sequence 2 (SEQ ID NO: 4)

Single underline is the exogenous signal peptide; the double underlined sequence part does not have furin tangent points, italics indicates the sequence design around furin tangent points. AMH sequence 2 cannot be cleaved by furin, thereby forming ProAMH.

WT sequencehttps://www.uniprot.org/uniprot/P03971

PTM/processing

Molecular processing

Feature keywords	Position of	Description of the invention
			Signal peptides	1-18	Sequence analysis
Propeptide (PRO_ 0000033746)	19-25	Sequence divisionAnalysis
			Chain ⁱ (PRO_0000033747)	26-560	Lees' tube inhibitor

AMH sequence 1 Gene sequence (SEQ ID NO: 1):

ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTCCTGCTGCTGTGGCCCATGGTGTGGGCCCACCACCACCACCATCACCACCACAGAGCTGAAGAACCCGCTGTGGGAACATCTGGCCTGATCTTCAGAGAGGACCTGGATTGGCCTCCTGGCAGCCCTCAAGAACCTCTGTGTCTGGTTGCTCTGGGCGGAGATAGCAATGGAAGCAGCAGCCCTCTTAGAGTGGTTGGAGCTCTGAGCGCTTACGAACAGGCTTTTCTGGGAGCCGTGCAAAGAGCTAGATGGGGCCCTAGAGACCTGGCTACCTTCGGCGTTTGTAACACCGGCGATAGACAGGCTGCTCTGCCTAGCCTTAGAAGACTGGGCGCTTGGCTGAGAGATCCTGGAGGACAGAGACTGGTGGTGCTGCATCTGGAAGAGGTGACCTGGGAACCTACCCCTAGCCTGAGATTTCAAGAGCCTCCTCCTGGAGGCGCTGGACCTCCTGAACTGGCTCTGCTGGTGCTGTATCCTGGACCTGGCCCTGAAGTGACAGTGACAAGAGCTGGACTGCCTGGAGCTCAAAGCCTGTGTCCCAGCAGAGACACCAGATACCTGGTGCTGGCTGTGGATAGACCTGCCGGAGCTTGGAGAGGATCTGGACTGGCTCTTACACTGCAACCCAGAGGCGAGGATTCTAGACTGAGCACCGCCAGACTGCAAGCTCTGCTGTTCGGCGATGACCACAGATGCTTCACCAGAATGACCCCTGCCCTGCTGCTTCTTCCTAGAAGCGAGCCTGCCCCTCTTCCTGCTCATGGACAGCTTGACACAGTGCCCTTTCCTCCTCCTAGACCCAGCGCTGAACTGGAAGAAAGCCCCCCTAGCGCTGATCCCTTTCTGGAGACCCTGACCAGACTTGTGAGAGCCCTGAGAGTGCCTCCTGCTAGAGCTAGCGCTCCTAGACTGGCTCTGGATCCCGATGCTCTGGCTGGATTCCCTCAAGGACTGGTGAACCTGTCTGATCCCGCCGCTCTGGAAAGACTGCTGGATGGCGAAGAACCTCTGCTGCTGCTGCTTAGACCTACAGCTGCTACCACAGGAGATCCCGCCCCTCTGCATGATCCTACAAGCGCCCCTTGGGCTACAGCTCTGGCTAGAAGAGTGGCTGCCGAACTGCAGGCTGCCGCTGCTGAACTTAGAAGCCTGCCTGGACTGCCTCCTGCTACAGCCCCTCTGCTTGCTAGACTGCTGGCTCTGTGTCCTGGCGGACCTGGAGGACTGGGAGATCCTCTGAGAGCTCTGCTGCTGCTGAAAGCTCTGCAAGGCCTGAGAGTGGAGTGGAGAGGCAGAGATCACCTGCAGAGCTCCAGGCACAGGAGGGCCCTGGATAGCGCCGGCGCTACAGCCGCTGACGGCCCTTGTGCCCTGAGGGAGCTGTCCGTGGATCTGAGGGCCGAGAGATCCGTGCTGATCCCTGAGACCTACCAGGCCAACAATTGCCAGGGCGTGTGCGGCTGGCCTCAGTCCGACAGGAACCCTAGATACGGCAACCACGTGGTGCTGCTGCTGAAGATGCAGGTGAGAGGCGCCGCCCTGGCCAGGCCTCCTTGTTGTGTGCCTACAGCCTACGCCGGCAAGCTGCTGATCTCCCTGAGCGAGGAGAGAATCTCCGCCCACCACGTGCCCAACATGGTGGCCACCGAGTGTGGCTGTAGATGA。

AMH sequence 2 Gene sequence (SEQ ID NO: 2):

ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTCCTGCTGCTGTGGCCCATGGTGTGGGCCCACCACCACCACCATCACCACCACAGAGCTGAAGAACCCGCTGTGGGAACATCTGGCCTGATCTTCAGAGAGGACCTGGATTGGCCTCCTGGCAGCCCTCAAGAACCTCTGTGTCTGGTTGCTCTGGGCGGAGATAGCAATGGAAGCAGCAGCCCTCTTAGAGTGGTTGGAGCTCTGAGCGCTTACGAACAGGCTTTTCTGGGAGCCGTGCAAAGAGCTAGATGGGGCCCTAGAGACCTGGCTACCTTCGGCGTTTGTAACACCGGCGATAGACAGGCTGCTCTGCCTAGCCTTAGAAGACTGGGCGCTTGGCTGAGAGATCCTGGAGGACAGAGACTGGTGGTGCTGCATCTGGAAGAGGTGACCTGGGAACCTACCCCTAGCCTGAGATTTCAAGAGCCTCCTCCTGGAGGCGCTGGACCTCCTGAACTGGCTCTGCTGGTGCTGTATCCTGGACCTGGCCCTGAAGTGACAGTGACAAGAGCTGGACTGCCTGGAGCTCAAAGCCTGTGTCCCAGCAGAGACACCAGATACCTGGTGCTGGCTGTGGATAGACCTGCCGGAGCTTGGAGAGGATCTGGACTGGCTCTTACACTGCAACCCAGAGGCGAGGATTCTAGACTGAGCACCGCCAGACTGCAAGCTCTGCTGTTCGGCGATGACCACAGATGCTTCACCAGAATGACCCCTGCCCTGCTGCTTCTTCCTAGAAGCGAGCCTGCCCCTCTTCCTGCTCATGGACAGCTTGACACAGTGCCCTTTCCTCCTCCTAGACCCAGCGCTGAACTGGAAGAAAGCCCCCCTAGCGCTGATCCCTTTCTGGAGACCCTGACCAGACTTGTGAGAGCCCTGAGAGTGCCTCCTGCTAGAGCTAGCGCTCCTAGACTGGCTCTGGATCCCGATGCTCTGGCTGGATTCCCTCAAGGACTGGTGAACCTGTCTGATCCCGCCGCTCTGGAAAGACTGCTGGATGGCGAAGAACCTCTGCTGCTGCTGCTTAGACCTACAGCTGCTACCACAGGAGATCCCGCCCCTCTGCATGATCCTACAAGCGCCCCTTGGGCTACAGCTCTGGCTAGAAGAGTGGCTGCCGAACTGCAGGCTGCCGCTGCTGAACTTAGAAGCCTGCCTGGACTGCCTCCTGCTACAGCCCCTCTGCTTGCTAGACTGCTGGCTCTGTGTCCTGGCGGACCTGGAGGACTGGGAGATCCTCTGAGAGCTCTGCTGCTGCTGAAAGCTCTGCAAGGCCTGAGAGTGGAGTGGAGAGGCAGAGATCACCTGCAGAGCGGAGGAGGAGGCGGAGCCCTGGATAGCGCCGGCGCTACAGCCGCTGACGGCCCTTGTGCCCTGAGGGAGCTGTCCGTGGATCTGAGGGCCGAGAGATCCGTGCTGATCCCTGAGACCTACCAGGCCAACAATTGCCAGGGCGTGTGCGGCTGGCCTCAGTCCGACAGGAACCCTAGATACGGCAACCACGTGGTGCTGCTGCTGAAGATGCAGGTGAGAGGCGCCGCCCTGGCCAGGCCTCCTTGTTGTGTGCCTACAGCCTACGCCGGCAAGCTGCTGATCTCCCTGAGCGAGGAGAGAATCTCCGCCCACCACGTGCCCAACATGGTGGCCACCGAGTGTGGCTGTAGATGA。

by now it should be appreciated by those skilled in the art that while a number of exemplary embodiments of the invention have been shown and described herein in detail, many other variations or modifications of the invention consistent with the principles of the invention may be directly ascertained or inferred from the present disclosure without departing from the spirit and scope of the invention. Accordingly, the scope of the present invention should be understood and deemed to cover all such other variations or modifications.

Sequence listing

<110> biotechnology (Shanghai) Co., ltd

<120> an engineered AMH gene sequence and method for preparing AMH using the same

<130> L20090305F

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1704

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atgtggtgga gactgtggtg gctgctgctg ctgctcctgc tgctgtggcc catggtgtgg 60

gcccaccacc accaccatca ccaccacaga gctgaagaac ccgctgtggg aacatctggc 120

ctgatcttca gagaggacct ggattggcct cctggcagcc ctcaagaacc tctgtgtctg 180

gttgctctgg gcggagatag caatggaagc agcagccctc ttagagtggt tggagctctg 240

agcgcttacg aacaggcttt tctgggagcc gtgcaaagag ctagatgggg ccctagagac 300

ctggctacct tcggcgtttg taacaccggc gatagacagg ctgctctgcc tagccttaga 360

agactgggcg cttggctgag agatcctgga ggacagagac tggtggtgct gcatctggaa 420

gaggtgacct gggaacctac ccctagcctg agatttcaag agcctcctcc tggaggcgct 480

ggacctcctg aactggctct gctggtgctg tatcctggac ctggccctga agtgacagtg 540

acaagagctg gactgcctgg agctcaaagc ctgtgtccca gcagagacac cagatacctg 600

gtgctggctg tggatagacc tgccggagct tggagaggat ctggactggc tcttacactg 660

caacccagag gcgaggattc tagactgagc accgccagac tgcaagctct gctgttcggc 720

gatgaccaca gatgcttcac cagaatgacc cctgccctgc tgcttcttcc tagaagcgag 780

cctgcccctc ttcctgctca tggacagctt gacacagtgc cctttcctcc tcctagaccc 840

agcgctgaac tggaagaaag cccccctagc gctgatccct ttctggagac cctgaccaga 900

cttgtgagag ccctgagagt gcctcctgct agagctagcg ctcctagact ggctctggat 960

cccgatgctc tggctggatt ccctcaagga ctggtgaacc tgtctgatcc cgccgctctg 1020

gaaagactgc tggatggcga agaacctctg ctgctgctgc ttagacctac agctgctacc 1080

acaggagatc ccgcccctct gcatgatcct acaagcgccc cttgggctac agctctggct 1140

agaagagtgg ctgccgaact gcaggctgcc gctgctgaac ttagaagcct gcctggactg 1200

cctcctgcta cagcccctct gcttgctaga ctgctggctc tgtgtcctgg cggacctgga 1260

ggactgggag atcctctgag agctctgctg ctgctgaaag ctctgcaagg cctgagagtg 1320

gagtggagag gcagagatca cctgcagagc tccaggcaca ggagggccct ggatagcgcc 1380

ggcgctacag ccgctgacgg cccttgtgcc ctgagggagc tgtccgtgga tctgagggcc 1440

gagagatccg tgctgatccc tgagacctac caggccaaca attgccaggg cgtgtgcggc 1500

tggcctcagt ccgacaggaa ccctagatac ggcaaccacg tggtgctgct gctgaagatg 1560

caggtgagag gcgccgccct ggccaggcct ccttgttgtg tgcctacagc ctacgccggc 1620

aagctgctga tctccctgag cgaggagaga atctccgccc accacgtgcc caacatggtg 1680

gccaccgagt gtggctgtag atga 1704

<210> 2

<211> 1704

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atgtggtgga gactgtggtg gctgctgctg ctgctcctgc tgctgtggcc catggtgtgg 60

gcccaccacc accaccatca ccaccacaga gctgaagaac ccgctgtggg aacatctggc 120

ctgatcttca gagaggacct ggattggcct cctggcagcc ctcaagaacc tctgtgtctg 180

gttgctctgg gcggagatag caatggaagc agcagccctc ttagagtggt tggagctctg 240

agcgcttacg aacaggcttt tctgggagcc gtgcaaagag ctagatgggg ccctagagac 300

ctggctacct tcggcgtttg taacaccggc gatagacagg ctgctctgcc tagccttaga 360

agactgggcg cttggctgag agatcctgga ggacagagac tggtggtgct gcatctggaa 420

gaggtgacct gggaacctac ccctagcctg agatttcaag agcctcctcc tggaggcgct 480

ggacctcctg aactggctct gctggtgctg tatcctggac ctggccctga agtgacagtg 540

acaagagctg gactgcctgg agctcaaagc ctgtgtccca gcagagacac cagatacctg 600

gtgctggctg tggatagacc tgccggagct tggagaggat ctggactggc tcttacactg 660

caacccagag gcgaggattc tagactgagc accgccagac tgcaagctct gctgttcggc 720

gatgaccaca gatgcttcac cagaatgacc cctgccctgc tgcttcttcc tagaagcgag 780

cctgcccctc ttcctgctca tggacagctt gacacagtgc cctttcctcc tcctagaccc 840

agcgctgaac tggaagaaag cccccctagc gctgatccct ttctggagac cctgaccaga 900

cttgtgagag ccctgagagt gcctcctgct agagctagcg ctcctagact ggctctggat 960

cccgatgctc tggctggatt ccctcaagga ctggtgaacc tgtctgatcc cgccgctctg 1020

gaaagactgc tggatggcga agaacctctg ctgctgctgc ttagacctac agctgctacc 1080

acaggagatc ccgcccctct gcatgatcct acaagcgccc cttgggctac agctctggct 1140

agaagagtgg ctgccgaact gcaggctgcc gctgctgaac ttagaagcct gcctggactg 1200

cctcctgcta cagcccctct gcttgctaga ctgctggctc tgtgtcctgg cggacctgga 1260

ggactgggag atcctctgag agctctgctg ctgctgaaag ctctgcaagg cctgagagtg 1320

gagtggagag gcagagatca cctgcagagc ggaggaggag gcggagccct ggatagcgcc 1380

ggcgctacag ccgctgacgg cccttgtgcc ctgagggagc tgtccgtgga tctgagggcc 1440

gagagatccg tgctgatccc tgagacctac caggccaaca attgccaggg cgtgtgcggc 1500

tggcctcagt ccgacaggaa ccctagatac ggcaaccacg tggtgctgct gctgaagatg 1560

caggtgagag gcgccgccct ggccaggcct ccttgttgtg tgcctacagc ctacgccggc 1620

aagctgctga tctccctgag cgaggagaga atctccgccc accacgtgcc caacatggtg 1680

gccaccgagt gtggctgtag atga 1704

<210> 3

<211> 567

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp

1 5 10 15

Pro Met Val Trp Ala His His His His His His His His Arg Ala Glu

20 25 30

Glu Pro Ala Val Gly Thr Ser Gly Leu Ile Phe Arg Glu Asp Leu Asp

35 40 45

Trp Pro Pro Gly Ser Pro Gln Glu Pro Leu Cys Leu Val Ala Leu Gly

50 55 60

Gly Asp Ser Asn Gly Ser Ser Ser Pro Leu Arg Val Val Gly Ala Leu

65 70 75 80

Ser Ala Tyr Glu Gln Ala Phe Leu Gly Ala Val Gln Arg Ala Arg Trp

85 90 95

Gly Pro Arg Asp Leu Ala Thr Phe Gly Val Cys Asn Thr Gly Asp Arg

100 105 110

Gln Ala Ala Leu Pro Ser Leu Arg Arg Leu Gly Ala Trp Leu Arg Asp

115 120 125

Pro Gly Gly Gln Arg Leu Val Val Leu His Leu Glu Glu Val Thr Trp

130 135 140

Glu Pro Thr Pro Ser Leu Arg Phe Gln Glu Pro Pro Pro Gly Gly Ala

145 150 155 160

Gly Pro Pro Glu Leu Ala Leu Leu Val Leu Tyr Pro Gly Pro Gly Pro

165 170 175

Glu Val Thr Val Thr Arg Ala Gly Leu Pro Gly Ala Gln Ser Leu Cys

180 185 190

Pro Ser Arg Asp Thr Arg Tyr Leu Val Leu Ala Val Asp Arg Pro Ala

195 200 205

Gly Ala Trp Arg Gly Ser Gly Leu Ala Leu Thr Leu Gln Pro Arg Gly

210 215 220

Glu Asp Ser Arg Leu Ser Thr Ala Arg Leu Gln Ala Leu Leu Phe Gly

225 230 235 240

Asp Asp His Arg Cys Phe Thr Arg Met Thr Pro Ala Leu Leu Leu Leu

245 250 255

Pro Arg Ser Glu Pro Ala Pro Leu Pro Ala His Gly Gln Leu Asp Thr

260 265 270

Val Pro Phe Pro Pro Pro Arg Pro Ser Ala Glu Leu Glu Glu Ser Pro

275 280 285

Pro Ser Ala Asp Pro Phe Leu Glu Thr Leu Thr Arg Leu Val Arg Ala

290 295 300

Leu Arg Val Pro Pro Ala Arg Ala Ser Ala Pro Arg Leu Ala Leu Asp

305 310 315 320

Pro Asp Ala Leu Ala Gly Phe Pro Gln Gly Leu Val Asn Leu Ser Asp

325 330 335

Pro Ala Ala Leu Glu Arg Leu Leu Asp Gly Glu Glu Pro Leu Leu Leu

340 345 350

Leu Leu Arg Pro Thr Ala Ala Thr Thr Gly Asp Pro Ala Pro Leu His

355 360 365

Asp Pro Thr Ser Ala Pro Trp Ala Thr Ala Leu Ala Arg Arg Val Ala

370 375 380

Ala Glu Leu Gln Ala Ala Ala Ala Glu Leu Arg Ser Leu Pro Gly Leu

385 390 395 400

Pro Pro Ala Thr Ala Pro Leu Leu Ala Arg Leu Leu Ala Leu Cys Pro

405 410 415

Gly Gly Pro Gly Gly Leu Gly Asp Pro Leu Arg Ala Leu Leu Leu Leu

420 425 430

Lys Ala Leu Gln Gly Leu Arg Val Glu Trp Arg Gly Arg Asp His Leu

435 440 445

Gln Ser Ser Arg His Arg Arg Ala Leu Asp Ser Ala Gly Ala Thr Ala

450 455 460

Ala Asp Gly Pro Cys Ala Leu Arg Glu Leu Ser Val Asp Leu Arg Ala

465 470 475 480

Glu Arg Ser Val Leu Ile Pro Glu Thr Tyr Gln Ala Asn Asn Cys Gln

485 490 495

Gly Val Cys Gly Trp Pro Gln Ser Asp Arg Asn Pro Arg Tyr Gly Asn

500 505 510

His Val Val Leu Leu Leu Lys Met Gln Val Arg Gly Ala Ala Leu Ala

515 520 525

Arg Pro Pro Cys Cys Val Pro Thr Ala Tyr Ala Gly Lys Leu Leu Ile

530 535 540

Ser Leu Ser Glu Glu Arg Ile Ser Ala His His Val Pro Asn Met Val

545 550 555 560

Ala Thr Glu Cys Gly Cys Arg

565

<210> 4

<211> 567

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp

1 5 10 15

Pro Met Val Trp Ala His His His His His His His His Arg Ala Glu

20 25 30

Glu Pro Ala Val Gly Thr Ser Gly Leu Ile Phe Arg Glu Asp Leu Asp

35 40 45

Trp Pro Pro Gly Ser Pro Gln Glu Pro Leu Cys Leu Val Ala Leu Gly

50 55 60

Gly Asp Ser Asn Gly Ser Ser Ser Pro Leu Arg Val Val Gly Ala Leu

65 70 75 80

Ser Ala Tyr Glu Gln Ala Phe Leu Gly Ala Val Gln Arg Ala Arg Trp

85 90 95

Gly Pro Arg Asp Leu Ala Thr Phe Gly Val Cys Asn Thr Gly Asp Arg

100 105 110

Gln Ala Ala Leu Pro Ser Leu Arg Arg Leu Gly Ala Trp Leu Arg Asp

115 120 125

Pro Gly Gly Gln Arg Leu Val Val Leu His Leu Glu Glu Val Thr Trp

130 135 140

Glu Pro Thr Pro Ser Leu Arg Phe Gln Glu Pro Pro Pro Gly Gly Ala

145 150 155 160

Gly Pro Pro Glu Leu Ala Leu Leu Val Leu Tyr Pro Gly Pro Gly Pro

165 170 175

Glu Val Thr Val Thr Arg Ala Gly Leu Pro Gly Ala Gln Ser Leu Cys

180 185 190

Pro Ser Arg Asp Thr Arg Tyr Leu Val Leu Ala Val Asp Arg Pro Ala

195 200 205

Gly Ala Trp Arg Gly Ser Gly Leu Ala Leu Thr Leu Gln Pro Arg Gly

210 215 220

Glu Asp Ser Arg Leu Ser Thr Ala Arg Leu Gln Ala Leu Leu Phe Gly

225 230 235 240

Asp Asp His Arg Cys Phe Thr Arg Met Thr Pro Ala Leu Leu Leu Leu

245 250 255

Pro Arg Ser Glu Pro Ala Pro Leu Pro Ala His Gly Gln Leu Asp Thr

260 265 270

Val Pro Phe Pro Pro Pro Arg Pro Ser Ala Glu Leu Glu Glu Ser Pro

275 280 285

Pro Ser Ala Asp Pro Phe Leu Glu Thr Leu Thr Arg Leu Val Arg Ala

290 295 300

Leu Arg Val Pro Pro Ala Arg Ala Ser Ala Pro Arg Leu Ala Leu Asp

305 310 315 320

Pro Asp Ala Leu Ala Gly Phe Pro Gln Gly Leu Val Asn Leu Ser Asp

325 330 335

Pro Ala Ala Leu Glu Arg Leu Leu Asp Gly Glu Glu Pro Leu Leu Leu

340 345 350

Leu Leu Arg Pro Thr Ala Ala Thr Thr Gly Asp Pro Ala Pro Leu His

355 360 365

Asp Pro Thr Ser Ala Pro Trp Ala Thr Ala Leu Ala Arg Arg Val Ala

370 375 380

Ala Glu Leu Gln Ala Ala Ala Ala Glu Leu Arg Ser Leu Pro Gly Leu

385 390 395 400

Pro Pro Ala Thr Ala Pro Leu Leu Ala Arg Leu Leu Ala Leu Cys Pro

405 410 415

Gly Gly Pro Gly Gly Leu Gly Asp Pro Leu Arg Ala Leu Leu Leu Leu

420 425 430

Lys Ala Leu Gln Gly Leu Arg Val Glu Trp Arg Gly Arg Asp His Leu

435 440 445

Gln Ser Gly Gly Gly Gly Gly Ala Leu Asp Ser Ala Gly Ala Thr Ala

450 455 460

Ala Asp Gly Pro Cys Ala Leu Arg Glu Leu Ser Val Asp Leu Arg Ala

465 470 475 480

Glu Arg Ser Val Leu Ile Pro Glu Thr Tyr Gln Ala Asn Asn Cys Gln

485 490 495

Gly Val Cys Gly Trp Pro Gln Ser Asp Arg Asn Pro Arg Tyr Gly Asn

500 505 510

His Val Val Leu Leu Leu Lys Met Gln Val Arg Gly Ala Ala Leu Ala

515 520 525

Arg Pro Pro Cys Cys Val Pro Thr Ala Tyr Ala Gly Lys Leu Leu Ile

530 535 540

Ser Leu Ser Glu Glu Arg Ile Ser Ala His His Val Pro Asn Met Val

545 550 555 560

Ala Thr Glu Cys Gly Cys Arg

565

Claims

1. A modified AMH gene, characterized in that: the sequence of the modified AMH gene is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

2. An engineered AMH protein, characterized in that: the amino acid sequence of the modified AMH protein is shown as SEQ ID NO. 3 or SEQ ID NO. 4.

3. An expression vector, characterized in that: the expression vector comprises the AMH gene of claim 1.

4. The expression vector of claim 3, wherein: the expression vector is a mammalian cell expression vector.

5. An AMH gene according to claim 1 for use in the preparation of AMH _N,C And the use of Pro-AMH proteins.

6. Preparation of AMH _N,C And a method for Pro-AMH protein, characterized in that: the method may include the steps of,

s1: constructing an AMH gene and a receptor protein AMHRII gene as claimed in claim 1 into a mammalian cell expression vector to form an AMH expression plasmid and an AMH RII-Fc expression plasmid respectively;

s2: co-transfecting the AMH expression plasmid prepared in the step S1 and the AMH RII-Fc expression plasmid into mammalian cells;

s3: s2 the mammalian cells express AMH _N,C Pro-AMH protein;

the mammalian cell expression vectors described in S1 include pTT5, pCDNA3.1, pCMV expression vectors;

the AMH expression plasmid and AMH RII-Fc expression plasmid described in S2 were used in the following manner: 1, co-transfection in proportion;

the mammalian cells in S2 are selected from the group consisting of an Expi293 cell or a CHO cell.