CN1259574A - New human phosphatide transferase, its code sequence, prepn. and use thereof - Google Patents

Abstract

The invention relates to a novel human phospholipid flippase PLS2. The invention provides the cDNA coding sequence of the new phospholipid flippase, the polypeptide coded by the sequence, and the method for producing the new human phospholipid flippase by recombinant technology. The invention also provides the application of the novel human phospholipid flippase and its coding sequence.

Description

Novel human phospholipid flippase, its coding sequence, production method and application

The invention relates to the field of genetic engineering. Specifically, the present invention relates to a novel polynucleotide, a polypeptide encoded by the polynucleotide, applications of these polynucleotides and polypeptides, and methods for producing the polynucleotides and the polypeptides. The polypeptide of the present invention is deduced and identified as a novel human phospholipid flippase.

The distribution of phospholipids on the plasma membrane is usually asymmetrical, with phosphatidylcholine and sphingomyelin generally distributed on the outside of the bilayer membrane, while aminophospholipids, phosphatidylserine and phosphatidic ethanol are confined to the cytoplasmic side (Schrait AJ et al Biochim. Biophys. Acta 1071:313-329, 1991). Phospholipid molecules generally seldom flip-flop in the plasma membrane, but the increase in intracellular Ca ²⁺ levels caused by physiological activities such as cell activation, cell injury, and apoptosis can trigger bidirectional movement of phospholipid molecules in the plasma membrane (Williamson P. et al Biochemistry 31:6355-6360, 1992). The turnover of phospholipid molecules on both sides of the membrane can promote the aggregation and activation of key enzymes in the coagulation system and complement system, and accelerate the removal of damaged cells and apoptotic cells (Bevers EM et al Blood Rev. 5:146-154, 1991).

Phospholipid scramblase (abbreviated as "PLS") is a newly discovered protein in recent years that can mediate Ca ²⁺ -dependent phospholipid molecular turnover. In 1996, Basse et al. isolated a membrane-integrated protein of about 37KD from human erythrocytes, which has the function of mediating the turnover of artificial liposome phospholipid molecules (Basse F.et al J.Biol.Chem.271( 29): 17205-17210, 1996); in 1997, the laboratory Zhou et al. cloned the cDNA sequence of the protein (Zhou Q. et al J. Biol. Chem. 272(29): 18240-18244, 1997); In 1998, Zhou et al. cloned the cDNA sequence of the homologue of this protein in mice (Zhou Q. et al Biochemistry 37: 2356-2360, 1998).

However, the novel human phospholipid flippase or its sequence involved in the present invention has not been disclosed before this application.

An object of the present invention is to provide a novel polynucleotide encoding a novel phospholipid flippase, and the novel human phospholipid flippase of the present invention is named as human PLS2.

Another object of the present invention is to provide a novel human phospholipid flippase protein, which is named as human PLS2.

Another object of the present invention is to provide a method for producing the novel human phospholipid flippase by recombinant technology.

The present invention also relates to the application of the human phospholipid flippase nucleic acid sequence and polypeptide.

In one aspect of the present invention, an isolated DNA molecule is provided, which includes: a nucleotide sequence encoding a polypeptide having human PLS2 protein activity, said nucleotide sequence is identical to that of SEQ ID NO. The nucleotide sequence at nucleotide 118-849 has at least 70% homology; or the nucleotide sequence can be obtained from nucleotide 118-849 in SEQ ID NO.3 under moderately stringent conditions nucleotide sequence hybridization. Preferably, said sequence encodes a polypeptide having the sequence shown in SEQ ID NO.4. More preferably, the sequence has the nucleotide sequence from nucleotide 118-849 in SEQ ID NO.3.

In another aspect of the present invention, an isolated human PLS2 protein polypeptide is provided, which includes: a polypeptide having the amino acid sequence of SEQID NO.4, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof. Preferably, the polypeptide is a polypeptide having the sequence of SEQ ID NO.4.

In another aspect of the present invention, there is provided a vector comprising the above isolated DNA.

In another aspect of the present invention, a host cell transformed with the vector is provided.

In another aspect of the present invention, a method for producing a polypeptide having human PLS2 protein activity is provided, the method comprising:

(a) The nucleotide sequence encoding the polypeptide having human PLS2 protein activity is operably connected to the expression control sequence to form a human PLS2 protein expression vector, and the nucleotide sequence and SEQ ID NO.3 are derived from nucleosides The nucleotide sequence of acid 118-849 has at least 70% homology;

(b) transferring the expression vector in step (a) into a host cell to form a recombinant cell of human PLS2 protein;

(c) cultivating the recombinant cells in step (b) under conditions suitable for expressing the human PLS2 protein polypeptide;

(d) isolating a polypeptide having human PLS2 protein activity.

In a specific embodiment of the present invention, the isolated polynucleotide of the present invention has a full length of 892 nucleotides, and its detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at 118-849 nucleotides.

In the present invention, "isolated", "purified" or "substantially pure" DNA means that the DNA or fragment has been separated from the sequences flanking it in its natural state, and also means that the DNA or fragment has been Separated from the components that naturally accompany nucleic acids and that have been separated from the proteins that accompany them in cells.

In the present invention, the term "human PLS2 protein (or polypeptide) coding sequence" refers to a nucleotide sequence encoding a polypeptide having human PLS2 protein activity, such as the 118-849 nucleotide sequence and its abbreviation in SEQ ID NO.3 and sequence. The degenerate sequence refers to a sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the 118-849 nucleotides of the coding frame of the sequence of SEQ ID NO.3. Due to the degeneracy of codons, a degenerate sequence with a homology as low as about 70% to the 118-849 nucleotide sequence in SEQ ID NO.3 can also encode the sequence described in SEQ ID NO.4. The term also includes a nucleotide sequence capable of hybridizing to the nucleotide sequence from nucleotides 118-849 in SEQ ID NO.3 under moderately stringent conditions, more preferably under highly stringent conditions. In addition, the term also includes at least 70%, preferably at least 80%, more preferably at least 90% of the nucleosides of the nucleotide sequence from nucleotide 118-849 in SEQ ID NO.3 acid sequence.

The term also includes variants of the open reading frame sequence of SEQ ID NO. 3 that encode a protein having the same function as human PLS2. These variations include (but are not limited to): the deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , insertion and/or substitution, and addition of several (usually within 60, preferably within 30, more preferably within 10, and most preferably within 5) at the 5' and/or 3' end ) nucleotides.

In the present invention, "substantially pure" protein or polypeptide means that it accounts for at least 20%, preferably at least 50%, more preferably at least 80%, and most preferably at least 90% (by dry weight) of the total substance of the sample. or wet weight). Purity can be measured by any suitable method, such as measuring the purity of the polypeptide by column chromatography, PAGE or HPLC. A substantially pure polypeptide is substantially free of components that accompany it in its native state.

In the present invention, the term "human PLS2 protein polypeptide" refers to a polypeptide having the sequence of SEQ ID NO.4 having human PLS2 protein activity. The term also includes variants of the sequence of SEQ ID NO.4 that have the same function as human phospholipid flippase PLS2. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the human PLS2 protein.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with human PLS2 DNA under high or low stringency conditions , and the polypeptide or protein obtained by using antiserum against human PLS2 polypeptide. The present invention also provides other polypeptides, such as fusion proteins comprising human PLS2 polypeptide or fragments thereof. In addition to substantially full-length polypeptides, the present invention also provides soluble fragments of human PLS2 polypeptides. Typically, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 80 contiguous amino acids of the human PLS2 polypeptide sequence. 100 consecutive amino acids.

The invention also provides analogs of human PLS2 protein or polypeptide. The difference between these analogues and the natural human PLS2 polypeptide may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs may also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, "human PLS2 conservative variant polypeptide" means that compared with the amino acid sequence of SEQ ID No.4, there are at most 10, preferably at most 8, and more preferably at most 5 amino acids that are similar or similar in nature. Amino acids are substituted to form polypeptides. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.

Table 1 initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

The present invention also includes antisense sequences of human PLS2 polypeptide coding sequences and fragments thereof. This antisense sequence can be used to inhibit the expression of human PLS2 in cells.

The present invention also includes a nucleotide molecule that can be used as a probe or a primer, which generally has 8-100, preferably 15-50 consecutive nucleotides of the human PLS2 nucleotide sequence. The probe can be used to detect whether there is a nucleic acid molecule encoding human PLS2 in a sample.

The present invention also includes a method for detecting human PLS2 nucleotide sequence, which comprises using the above-mentioned probe to hybridize with a sample, and then detecting whether the probe is combined. Preferably, the sample is a product of PCR amplification, wherein the PCR amplification primers correspond to the coding sequence of the human PLS2 polypeptide and can be located on both sides or in the middle of the coding sequence. Primers are generally 20-50 nucleotides in length.

In the present invention, various vectors known in the art, such as commercially available vectors, can be used. For example, a commercially available vector is selected, and then the nucleotide sequence encoding the polypeptide of the present invention is operably linked to the expression control sequence to form a protein expression vector.

As used herein, "operably linked" refers to the condition that some portion of a linear DNA sequence is capable of affecting the activity of other portions of the same linear DNA sequence. For example, a signal peptide (secretion leader) DNA is operably linked to a polypeptide DNA if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide; if a promoter controls the transcription of the sequence, it is operably linked to A coding sequence; a ribosome binding site is operably linked to a coding sequence if it is placed in a position to enable its translation. Generally, "operably linked to" means adjacent, and with respect to a secretory leader it means adjacent in reading frame.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cells are eukaryotic cells, such as CHO cells, COS cells and the like.

On the other hand, the present invention also includes antibodies specific to human PLS2 DNA or polypeptides encoded by its fragments, especially monoclonal antibodies. Here, "specificity" means that the antibody can bind to human PLS2 gene product or fragment. Preferably, it refers to those antibodies that can bind to human PLS2 gene products or fragments but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules capable of binding and inhibiting human PLS2 protein, as well as those antibodies that do not affect the function of human PLS2 protein. The invention also includes antibodies that bind to modified or unmodified forms of the human PLS2 gene product.

The present invention includes not only complete monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., US Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have the binding specificity of a murine antibody but retain portions of the antibody from humans.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, a purified human PLS2 gene product, or an antigenic fragment thereof, can be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing human PLS2 or antigenic fragments thereof can be used to immunize animals to produce antibodies. Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6: 511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981). The antibodies of the present invention include antibodies capable of blocking the function of human PLS2 and antibodies that do not affect the function of human PLS2. All kinds of antibodies of the present invention can be obtained by using fragments or functional regions of human PLS2 gene products through immunization techniques. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of the human PLS2 gene product can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g., E. coli); antibodies that bind to post-translationally modified forms (such as glycosylated or phosphorylated Proteins or polypeptides), which can be obtained by immunizing animals with gene products produced in eukaryotic cells (such as yeast or insect cells).

The full-length human PLS2 nucleotide sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the SEQ ID NO.3 sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a known method can be used as a template to amplify And get the relevant sequence. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Of course, fragments with very long sequences can be obtained by synthesizing multiple small fragments and then connecting them.

In one embodiment of the present invention, the full length of the polynucleotide of the present invention is 892 nucleotides, its detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at 118-849 nucleotides. The polynucleotide was obtained by using the human brain λgt11 cDNA library (purchased from Clontech) as a template to synthesize forward primer A1: 5'-TACAGTCCACAGCAACCCAGTAC-3' and reverse primer B1: 5'-AGTGCTGACTGTAAGCCCAATCC-3' PCR to obtain the target fragment of 892bp. After sequencing, the full-length cDNA sequence of SEQ ID NO.3 was obtained.

According to the result of homologous comparison, the nucleotide sequence of the present invention and its encoded protein sequence have shown significant homology with phospholipid flippase from different sources, therefore, this shows that it is a new homologue of phospholipid flippase , and has some important functions of phospholipid flippase proteins.

Phospholipid molecules on the plasma membrane are distributed asymmetrically (Schrait AJ et al Biochim. Biophys. Acta 1071: 313-329, 1991). Increased intracellular Ca ²⁺ levels can lead to rapid, bidirectional turnover of phospholipid molecules on the lipid bilayer cell membrane (Williamson P. et al Biochemistry 31: 6355-6360, 1992). Phospholipid flippase is widely expressed on the cell membranes of various cells and can mediate Ca ²⁺ -dependent protein molecules that move bidirectionally on cell membranes (Basse F.et al J.Biol.Chem.271(29): 17205-17210, 1996). The redistribution of phospholipid molecules in the cell membrane lipid bilayer molecules mediated by phospholipid flippase plays an important role in the mechanism of vascular hemostasis and cell clearance (Zhou Q. et al J. Biol. Chem. 272 (29): 18240-18244, 1997). The expression level of phospholipid flippase usually corresponds to the degree to which phosphatidylserine is flipped to the outside of the cell membrane in response to an increase in Ca ²⁺ levels (Zhou J. et al J. Biol. Chem. 273(12): 6603-6606, 1998). The homologue comparison of human and mouse shows that the cytoplasmic part of the protein contains a conserved Ca ²⁺ binding domain that is very similar to the known chiral structure sequence of EF (Zhou Q. et al Biochemistry37: 2356-2360, 1998), this structure is related to Ca ²⁺ regulating the activity of the enzyme (Zhou J. et al Biochemistry 37:6361-6366, 1998). Scott Syndrome is related to the loss of function of phospholipid flippase (Zhou Q. et al J. Biol. Chem. 272(29): 18240-18244, 1997). The human leukemogenesis-related gene MmTRA1b, which has an excellent match with the phospholipid flippase sequence, may be related to leukemogenesis and differentiation of monocytic leukemia cells (Kasukabe T. et al Biochem Biophys. Res. Commun. 249 (2): 449 -455, 1998).

In the attached picture,

Fig. 1 is a homologous comparison diagram of the nucleic acid sequences of human PLS2 and mouse phospholipid flippase (MPLS) of the present invention. Among them, the same nucleotides are marked with "|".

Fig. 2 is a homology comparison diagram of the amino acid sequences of human PLS2 and mouse phospholipid flippase (MPLS) of the present invention. Among them, the same amino acid is marked with "|" between the two sequences, and the similar amino acid is marked with "·". Similar amino acids are: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Fig. 3 is a homology comparison diagram of the nucleic acid sequences of human PLS2 and human phospholipid flippase (HPLS) of the present invention. Among them, the same nucleotides are marked with "|".

Fig. 4 is a homologous comparison diagram of the amino acid sequences of human PLS2 and human phospholipid flippase (HPLS) of the present invention. Among them, the same amino acid is marked with "|" between the two sequences, and the similar amino acid is marked with "·". Similar amino acids are: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Fig. 5 is a homology comparison diagram of the amino acid sequences of human PLS2 of the present invention and human leukemogenesis-related gene MmTRA1b.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Example 1

Cloning and Determination of the cDNA Sequence of Human PLS2

1. Primer amplification

Human brain λgt11 cDNA library (purchased from Clontech) was used as a template, and a pair of oligonucleotides were used as primers——A1: 5′-TACAGTCCACAAGCAACCCAGTAC-3′ (SEQ ID NO.1) was used as a forward primer, and the oligonucleotide B1: 5'-AGTGCTGACTGTAAGCCCAATCC-3' (SEQ ID NO.2) was used as a reverse primer for PCR. The PCR condition was 93°C for 4 minutes, followed by 35 cycles of 93°C for 1 minute, 68°C for 1 minute and 72°C for 1 minute, and finally 72°C for 5 minutes. The PCR fragment obtained by electrophoresis detection is about 900bp target fragment.

2. Sequencing of PCR products

The PCR amplified product obtained as above was ligated with the pGEM- ^T vector (Promega), transformed Escherichia coli JM103, extracted the plasmid with the QIAprep Plasmid kit (QIAGEN), and paired the insert with the double-stranded nested deletion kit (Pharmacia). Perform directional serial deletions, and then use PCR to quickly identify and sort the deletions. The sequentially truncated deletions were sequenced using the SequiTherm EXCEL ^TM DNA Sequencing Kit (Epicentre Technologies), and finally the sequence was spliced using computer software to obtain the full-length cDNA sequence, a total of 892bp. For the detailed sequence, see SEQ ID NO.3, where the open read Boxes are located at nucleotides 118-849.

According to the obtained full-length cDNA sequence, the amino acid sequence of human PLS2 was deduced, with a total of 243 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.4.

Example 2

Homology comparison and functional studies

Using the full-length cDNA sequence of human PLS2 of the present invention and its encoded protein, in the Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS translations+PDB+SwissProt+Spupdate+PIR database, use BLAST software to perform nucleic acid And protein homology search, it was found that it showed high homology with phospholipid flippase. Analyzed by PCGENE software, it has 70.3% identity (Figure 1) at the nucleic acid level and 48.6% identity at the protein level with mouse phospholipid flippase (MPLS) (gi|2935162|gb|AF015790|AF015790) identity and another 13.6% amino acid similarity (Figure 2); as another example, it has 70.3% identity with human phospholipid flippase (HPLS) (gi|2282600|gb|AF008445|AF008445) at the nucleic acid level ( Figure 3), there is 48.6% identity at the protein level and another 13.6% amino acid similarity (Figure 4). This shows that the novel protein of the present invention is a member of the phospholipid flippase family, and the function of the phospholipid flippase can be used to deduce the function of human PLS2.

Studies have shown that phospholipid molecules on the plasma membrane are distributed asymmetrically (Schrait AJ et al Biochim. Biophys. Acta 1071: 313-329, 1991). In quiescent cells, the turnover rate of phospholipid molecules spontaneously between the cell membrane bilayers is very slow, and the increase of intracellular Ca ²⁺ level caused by cell activation, cell injury, cell apoptosis, etc. can lead to phospholipid molecules in the lipid The rapid, bi-directional turnover on the double cell membrane makes the phosphatidylserine on the cytoplasmic side located on the outside, and the phosphatidylethanol on the outside is translocated to the cytoplasmic side (Williamson P. et al Biochemistry 31: 6355-6360, 1992) . Phospholipid flippase is a protein molecule widely expressed on the cell membrane of various cells, and it can mediate Ca ²⁺ dependent phospholipid molecules to move bidirectionally on the cell membrane (Basse F.et al J.Biol.Chem.271(29) : 17205-17210, 1996).

Studies have shown that phospholipid flippase is a cell membrane protein rich in proline type II (the protein of the present invention contains 15 prolines, accounting for more than 6% of the total length of the protein molecule), and has a penetrating protein near the C-terminus. Membrane region (Zhou Q. et al J. Biol. Chem. 272 (29): 18240-18244, 1997), the corresponding sequence of the present invention is the 204th to 224th amino acids in SEQ ID NO.4, namely LDVKMKAMIFGACFLIDFMYF. This further confirms that the novel protein of the present invention is a novel phospholipid flippase.

Experiments have shown that exposure of phospholipids to the outside can promote the aggregation and activation of key enzymes in the coagulation system and complement system, and accelerate the removal of damaged cells and apoptotic cells by the reticuloendothelial system (Bevers EMet alBlood Rev.5: 146- 154, 1991). Phospholipid flippase-mediated redistribution of Ca ²⁺ -dependent phospholipid molecules in the lipid bilayer of cell membrane plays an important role in vascular hemostasis mechanism and cell clearance mechanism (Zhou Q.et al J.Biol.Chem.272(29) : 18240-18244, 1997). Quantitative immunoblotting experiments show that the number of phospholipid flippase molecules in platelets is about 10 times higher than that of red blood cells, which is consistent with the significantly increased phospholipid flippase activity in platelet cell membranes (Zhou Q.et al J.Biol.Chem.272(29) : 18240-18244, 1997). The expression level of phospholipid flippase generally corresponds to the degree to which phosphatidylserine is flipped to the outside of the cell membrane as Ca ²⁺ levels rise (Zhou J. et al J. Biol. Chem. 273(12):6603-6606, 1998). Comparison of human, mouse and human homologues shows that the cytoplasmic part of the protein contains a conserved Ca ²⁺ binding domain that is very similar to the known EF chiral structure sequence:

D(A/S)DNFGIQFPL D

[Note: "(A/S)" in this sequence means that one amino acid is selected from these two amino acids] (Zhou Q. et al Biochemistry 37: 2356-2360, 1998), the corresponding sequence of the present invention is SEQ ID NO.4 Amino acids from the 192nd to the 203rd, namely DADHFDIHFPLD (the 197th and 199th amino acids are different from the corresponding amino acids of the reported phospholipid flippase, and the 195th amino acid is similar to the corresponding amino acid of the reported phospholipid flippase) , the structure is related to Ca ²⁺ regulating the activity of the enzyme. It was demonstrated experimentally that phospholipid flippase can be palmitoylated at the N-terminal cysteine residue remote from the Ca ²⁺ binding site. This post-translational modification may be related to the Ca ²⁺ -dependent activity of the enzyme (Zhou J. et al Biochemistry 37:6361-6366, 1998). The recombinant protein expressed in E. coli showed a significant decrease in both enzyme activity and Ca ²⁺ binding capacity. After hydrolysis of the thioether bond, the enzyme loses about 80% of its function (Zhou J. et al Biochemistry 37:6361-6366, 1998).

A genetic defect bleeding disease - Scott syndrome (Scott Syndrome) is related to the loss of function of phospholipid flippase (Zhou Q. et al J. Biol. Chem. 272 (29): 18240-18244, 1997). The human leukemogenesis-related gene MmTRA1b newly cloned by Kasukabe et al. has an excellent match with phospholipid flippase in amino acid sequence, so these proteins may be related to leukemogenesis and differentiation of monocytic leukemia cells (Kasukabe T. et al Biochem Biophys. Res. Commun. 249(2):449-455, 1998). The novel phospholipid flippase of the present invention also has high homology with MmTRA1b ( FIG. 5 ). This suggests that the novel protein of the present invention may also be related to leukemia.

In addition to being used as a member of the family for further functional research, the human PLS2 of the present invention can also be used to produce fusion proteins together with other proteins, such as producing fusion proteins together with immunoglobulins. In addition, human PLS2 of the present invention can also be fused or exchanged fragments with other members of the family to generate new proteins. For example, the N-terminal of human PLS2 of the present invention is exchanged with the N-terminal of mouse PLS or human PLS to produce new proteins with higher activity or new properties.

The antibody against human PLS2 of the present invention is used for screening other members of this family, or for affinity purification of related proteins (such as other members of this family, such as mouse PLS).

For example, the human PLS2 nucleic acid (coding sequence or antisense sequence) of the present invention can be introduced into cells to increase the expression level of human PLS2 or to inhibit the overexpression of human PLS2. The human PLS2 protein or its active polypeptide fragments of the present invention can be administered to patients to treat or alleviate related diseases caused by the lack, non-function or abnormality of human PLS2. In addition, the nucleic acid sequence or antibody based on the present invention can also be used for relevant diagnosis or prognosis.

Example 3

Expression of human PLS2 in Escherichia coli

The cDNA sequence encoding human PLS2 was amplified with PCR oligonucleotide primers corresponding to the 5' and 3' ends of the DNA sequence, using the fragment obtained in Example 1 as a template to synthesize an insert fragment.

The 5' oligonucleotide primer sequence is

5'-TTGCGGATCCATGCCAGGGCCAACTCCTAT-3' (SEQ ID NO.5),

The primer contains a BamHI restriction endonuclease cutting site, followed by 20 nucleotides of the human PLS2 coding sequence starting from the start codon;

The 3' oligonucleotide primer sequence is

5'-GTTCGTCGACTTAACCATAGGTGATGGCT-3' (SEQ ID NO.6),

The primer contains the cutting site of SalI restriction endonuclease, a translation terminator and the coding partial sequence of human PLS2.

The restriction enzyme cut site corresponds to the restriction endonuclease cut site on the bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, CA), which encodes antibiotic resistance (Amp ^r ) , a bacterial origin of replication (ori), an IPTG-regulated promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction Endonuclease cloning site.

The pQE-9 vector and the insert were digested with BamHI and SalI, and the insert was ligated into the pQE-9 vector keeping the open reading frame at the beginning of the bacterial RBS. The ligation mixture was subsequently used to transform an E. coli strain purchased from Qiagen under the tradename M15/rep4 containing multiple copies of the plasmid pREP4 expressing the lacI repressor and carrying kanamycin resistance (Kan ^r ). Transformants were screened on LB dishes containing Amp and Kan, and positive cells containing the desired construct were cultured overnight (O/N) in LB broth supplemented with Amp (100 μg/ml) and Kan (25 μg/ml) Transformant clones. The plasmid was extracted, and the results of sequencing verification showed that the cDNA insert of human PLS2 had been correctly loaded into the vector.

The overnight (O/N) culture was diluted at a dilution rate of 1:100-1:250, and then inoculated into a large volume of culture medium. When the cultured cells grew to 600 optical density (OD ₆₀₀ ) of 0.4-0.6, IPTG ( "Isopropylthio-β-D-galactoside") to a final concentration of 1 mM. By inactivating the lacI repressor, IPTG-induced initiation of P/O leads to increased gene expression levels. Cells were incubated for an additional 3-4 hours followed by centrifugation (6000 xg, 20 minutes). Inclusion bodies were lysed by sonication, cells were collected and the cell pellet was dissolved in 6M guanidine hydrochloride. After clarification, solubilized human PLS2 is purified from solution by nickel-chelate column chromatography under conditions that allow tight binding of the 6-His tag-containing protein. Human PLS2 was eluted from the column with 6M guanidine hydrochloride (pH 5.0). Several methods can be used to denature and precipitate proteins from guanidine hydrochloride. First, a dialysis step is used to remove the guanidine hydrochloride, or the purified protein isolated from the nickel-chelation column can be bound to a second column with a decreasing linear guanidine hydrochloride gradient. Proteins were denatured upon binding to the column and subsequently eluted with guanidine hydrochloride (pH 5.0). Finally, the soluble protein was dialyzed against PBS, and the protein was then stored in a stock solution with a final concentration of 10% (w/v) glycerol.

Electrophoresis was carried out with 12% SDS-PAGE gel, and the molecular weight of the expressed protein was identified as 28KDa.

In addition, the N-terminal and C-terminal 10 amino acid lengths of the expressed protein were sequenced by conventional methods, and it was found to be consistent with the sequence of SEQ ID NO.4.

Example 4

Expression of Human PLS2 in Eukaryotic Cells (CHO Cell Line)

In this example, the cDNA sequence encoding human PLS2 was amplified with PCR oligonucleotide primers corresponding to the 5' and 3' ends of the DNA sequence, using the fragment obtained in Example 1 as a template to synthesize the inserted fragment.

The 5' oligonucleotide primer sequence used in the PCR reaction is:

5'-TTGCAAGCTTATGCCAGGGCCAACTCCTAT-3' (SEQ ID NO.7),

The primer contains a HindIII restriction endonuclease cutting site, followed by 20 nucleotides of the human PLS2 coding sequence starting from the initiation codon;

The 3' end primer sequence is:

5'-GTTCGGATCCTTAACCATAGGTGATGGCT-3' (SEQ ID NO.8)

The primer contains the cutting site of BamHI restriction endonuclease, a translation terminator and the coding partial sequence of human PLS2.

The restriction endonuclease cutting sites on the primers correspond to the restriction endonuclease cutting sites on the CHO cell expression vector pcDNA3, which encodes antibiotic resistance (Amp ^r and Neo ^r ), a phage replication Origin (f1 ori), a viral origin of replication (SV40 ori), a T7 promoter, a viral promoter (P-CMV), a Sp6 promoter, an SV40 promoter, an SV40 tailing signal and the corresponding polyA sequence , a BGH tailed signal and the corresponding polyA sequence.

The pcDNA3 vector and the insert were digested with HindIII and BamHI, and then the insert was ligated into the pcDNA3 vector. The E. coli DH5α strain was subsequently transformed with the ligation mixture. Transformants were screened on LB dishes containing Amp, and clones containing the desired construct were cultured overnight (O/N) in LB liquid medium supplemented with Amp (100 μg/ml). The plasmid was extracted, and the results of sequencing verification showed that the cDNA insert of human PLS2 had been correctly loaded into the vector.

Plasmid transfection was performed by lipofection with Lipofectin kit (GiBco Life). After 48 hours of transfection, after 2-3 weeks of continuous G418 pressurized selection, the cells and cell supernatant were collected to measure the protease activity expressed. G418 was removed and subcultured continuously; for extreme dilution of mixed clone cells, select cell subclones with higher protein activity. The above-mentioned positive subclones were cultured in large quantities according to conventional methods. After 48 hours, the cells and supernatant were collected, and the cells were disrupted by ultrasonic lysis. With 50mM Tris HCl (pH 7.6) solution containing 0.05% Triton as the balance and eluent, the activity peaks of the above proteins were collected with a pre-balanced Superdex G-75 column. Then use a DEAE-Sepharose column equilibrated with 50mM Tris HCl (pH8.0) to carry out gradient elution with 50mM Tris HCl (pH8.0) solution containing 0-1M NaCl as the eluent, and collect the activity peak of the above protein. Then, the expressed protein solution was dialyzed with PBS (pH7.4) as the dialysate. Finally freeze-dried and stored.

Electrophoresis was carried out with 12% SDS-PAGE gel, and the molecular weight of the expressed protein was identified as 28KDa.

In addition, the N-terminal and C-terminal 10 amino acid lengths of the expressed protein were sequenced by conventional methods, and it was found to be consistent with the sequence of SEQ ID NO.4.

Example 5

Antibody preparation

The recombinant proteins obtained in Examples 3 and 4 were used to immunize animals to produce antibodies, as follows. The recombinant molecules are separated by chromatography for further use. It can also be separated by SDS-PAGE gel electrophoresis, and the electrophoresis bands are excised from the gel and emulsified with an equal volume of complete Freund's adjuvant. Mice were injected intraperitoneally with 50-100 [mu]g/0.2 ml emulsified protein. Fourteen days later, mice were boosted by intraperitoneal injection of the same antigen emulsified with incomplete Freund's adjuvant at a dose of 50-100 µg/0.2 ml. Give booster immunizations at least three times every 14 days. The specific reactivity of the obtained antiserum was assessed by its ability to precipitate the human PLS2 gene translation product in vitro. It was found that the antibody can specifically precipitate the protein of the present invention.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing (1) General Information: (i) Applicant: Fudan University (ii) Title of Invention: Novel Human Phospholipid Flippase, Its Coding Sequence, Preparation and Use (iii) Number of Sequences: 8 (2) SEQ ID NO .1 Information for (i) Sequential Features

(A) Length: 23 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topological structure: linear (ii) Molecular type: oligonucleotide (xi) Sequence description: SEQ ID NO.1: TACAGTCCAC AGCAACCCAG TAC 23 (2) Information of SEQ ID NO.2 (i) Sequence characteristics

(A) Length: 23 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topological structure: linear (ii) Molecular type: oligonucleotide (xi) Sequence description: SEQ ID NO: 2: AGTGCTGACT GTAAGCCCAA TCC 23 (2) Information of SEQ ID NO.3: (i) Sequence characteristics:

(A) Length: 892bp

(B) Type: nucleic acid

(C) chain: single chain

(D)拓扑结构：线性(ii)分子类型：cDNA(xi)序列描述：SEQ ID NO.3TACAGTCCAC  AGCAACCCAG  TACCTTCCCT  TTGTACCAGC  CAGTTGGTGG  TATCCATCCT  60GTCCGGTATC  AGCCTGGAAA  ATATCCTATG  CCAAATCAGT  CTGTTCCAAT  AACATGGATG  120CCAGGGCCAA  CTCCTATGGC  AAACTGCCCT  CCTGGTCTGG  AATACTTAGT  TCAGTTGGAC  180AACATACATG  TTCTTCAGCA  TTTTGAGCCT  CTGGAAATGA  TGACATGTTT  TGAAACTAAT  240AATAGATATG  ATATTAAAAA CAACTTAGAC  CAGATTGGTT  TTACATTTGT  AACCGAAGAC  300ACAGATGACG  GTTACCCGGG  AGGAACTGCC  TATCGGACAC  TAAGGCCCTT  CGGTCTCCGG  360GTCACTGATT  GTATGGGCCG  AGAAATCATG  ACAATGCAGA  GACCCTTCAG  ATGCACCTGC  420TGTTGCTTCT  GTTGCCCCTC  TGCCAGACAA  GAGCTGGAGG  TGCAGTGTCC  TCCTGGTGTC  480ACCATTGGCT  TTGTTGCGGA  ACATTGGAAC  CTGTGCAGGG  CGGTGTACAG  CATCCAAAAT  540GAGAAGAAAG  AAAATGTGAT  GAGAGTTCGT  GGGCCATGCT  CAACCTATGG  CTGTGGTTCA  600GATTCTGTTT  TTGAGGTCAA  ATCCCTTGAT  GGCATATCCA  ACATCGGCAG  TATTATCCGG  660AAGTGGAATG  GTTTGTTATC  AGCAATGGCA  GATGCTGACC  ATTTTGACAT  TCACTTCCCA  720CTAGACCTGG  ATGTGAAGAT  GAAAGCCATG  ATTTTTGGAG CTTGCTTCCT CATTGACTTC 780ATGTATTTTG AAAGATCTCC AACAACGTT CAAGATAGAG AGACACAGCA AGCCATCACC 840TATGGTTAAT TTTGAAAAAT GGAAAAGTTG GATTGGGCTT ACAGTCAGCA CT .NO 4Q ID: (

(A) Length: 243 amino acids

(B) Type: amino acid

(D) Topological structure: linear (ii) Molecular type: polypeptide (xi) Sequence description: SEQ ID NO.4: Met Pro Gly Pro Thr Pro Met Ala Asn Cys Pro Pro Gly Leu Glu 15Tyr Leu Val Gln Leu Asp Asn Ile His Val Leu Gln His Phe Glu 30Pro Leu Glu Met Met Thr Cys Phe Glu Thr Asn Asn Arg Tyr Asp 45Ile Lys Asn Asn Leu Asp Gln Ile Gly Phe Thr Phe Val Thr Glu 60Asp Thr Asp Asp Gly Tyr Pro Gly Gly Thr Ala Tyr Arg Thr Leu 75Arg Pro Phe Gly Leu Arg Val Thr Asp Cys Met Gly Arg Glu Ile 90Met Thr Met Gln Arg Pro Phe Arg Cys Thr Cys Cys Cys Phe Cys 105Cys Pro Ser Ala Arg Gln Glu Leu Glu Val Gln Cys Pro Pro Gly 120Val Thr Ile Gly PHE VAL Ala Glu His TRP Asn Leu Cys ARG ALA 135VAL TYR Serle Gln Glu LYS LYS GLU Asn Val Met ARG Val Gly Pro Cysr Tyr Tyr Tyr PHES SELUSLE 165GLU VAL LYS LYS LYS LYS LYS LYS Lys Asn Ile Gly Serle Ile Ile Ile Glu 225Arg Ser Pro His Asn Val Gln Asp Arg Glu Thr Gln Gln Ala Ile 240Thr Tyr Gly Information about sequence of features (2) SEQ ID NO.5

(A) Length: 30 bases

(B) Type: nucleic acid

(C) chain: single chain

(D) Topological structure: linear (ii) Molecular type: oligonucleotide (xi) Sequence description: SEQ ID NO.5: TTGCGGATCC ATGCCAGGGC CAACTCCTAT 30 (2) Information of SEQ ID NO.6 (i) Sequence characteristics

(A) Length: 29 bases

(B) Type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(xi) Sequence description: SEQ ID NO.6: GTTCGTCGAC TTAACCATAG GTGATGGCT 29 (2) Information of SEQ ID NO.7

(i) Sequential features

(A) Length: 30 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(xi) Sequence description: SEQ ID NO.7: TTGCAAGCTT ATGCCAGGGC CAACTCCTAT 30 (2) Information of SEQ ID NO.8

(i) Sequential features

(A) Length: 29 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(xi) Sequence description: SEQ ID NO.8: GTTCGGATCC TTAACCATAG GTGATGGCT 29

Claims (14)

1. An isolated DNA molecule, characterized in that it comprises: a nucleotide sequence encoding a polypeptide with human PLS2 protein activity, The nucleotide sequence has at least 70% homology to the nucleotide sequence from nucleotides 118 to 849 in SEQ ID NO.3; or The nucleotide sequence can hybridize with the nucleotide sequence from nucleotide 118-849 in SEQ ID NO.3 under moderately stringent conditions. 2. DNA molecules as claimed in claim 1, is characterized in that, described sequence codes a polypeptide, and this polypeptide has the sequence shown in SEQ ID NO.4. 3. DNA molecules as claimed in claim 1, is characterized in that, this sequence has the nucleotide sequence from nucleotide 118-849 in SEQ ID NO.3. 4. An isolated human PLS2 protein polypeptide, characterized in that it comprises: a polypeptide having the amino acid sequence of SEQ ID NO.4, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof. 5. The polypeptide according to claim 4, wherein the polypeptide is a polypeptide having a sequence of SEQ ID NO.4. 6. A vector comprising the DNA according to claim 1. 7. A host cell transformed with the vector according to claim 6. 8. The host cell of claim 7, wherein the cell is Escherichia coli. 9. The host cell of claim 7, which is a eukaryotic cell. 10. A method for producing a polypeptide having human PLS2 protein activity, characterized in that the method comprises: (a) The nucleotide sequence encoding the polypeptide having human PLS2 protein activity is operably connected to the expression control sequence to form a human PLS2 protein expression vector, and the nucleotide sequence and SEQ ID NO.3 are derived from nucleosides The nucleotide sequence of acid 118-849 has at least 70% homology; (b) transferring the expression vector in step (a) into a host cell to form a recombinant cell of human PLS2 protein; (c) culturing the recombinant cells in step (b) under conditions suitable for expressing the human PLS2 protein polypeptide; (d) isolating a polypeptide having human PLS2 protein activity. 11. The method of claim 10, wherein the sequence is from nucleotide 118-849 in SEQ ID NO.3. 12. An antibody capable of specifically binding to the human PLS2 protein polypeptide according to claim 4. 13. A nucleotide molecule, characterized in that it is the antisense sequence of the DNA molecule of claim 1. 14. A probe molecule, characterized in that it contains about 8-100 consecutive nucleotides in the DNA molecule of claim 1.

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