JP5029751B2 - Recombinant canine prolactin and method for producing the same - Google Patents

Description

  The present invention relates to elucidation of canine prolactin gene sequences and production of recombinant canine prolactin. By identifying the canine prolactin gene sequence, it is possible to produce canine prolactin using gene recombination technology, and accurately measure the blood concentration of prolactin, one of the factors involved in the incidence of breast cancer in dogs. It becomes possible.

  Peptide and protein hormones vary in structure among animal species and have a structure specific to animal species, and therefore, development of an animal species-specific measurement system is required for accurate measurement. However, a peptide / protein measurement system specific to companion animals is not yet established as compared to humans and cattle. In particular, it is desired to develop an accurate measurement system for hormones that can be used for diagnosis of diseases in companion animals.

  Normally, hormones in the blood are measured by an immunocompetitive method that uses affinity for antibodies, and there are RIA and ELISA methods depending on the detection method. When measuring a certain type of hormone in this way, it is necessary to use at least that standard (hormone) as the standard, because the affinity of the sample and standard for the antibody is different, so accurate measurements cannot be obtained. . Therefore, in order to obtain an accurate measurement value, it is necessary to use a species-specific measurement system using the hormone of the animal species.

  Prolactin (PRL) is a protein hormone secreted from the anterior pituitary gland and has many physiological functions involved in reproduction, behavior, growth / development, immunity and metabolism. In particular, it plays an important role in the differentiation and development of the mammary gland and promotes milk secretion. Currently, prolactin has been reported to increase breast cancer risk and has potential applications in breast cancer diagnosis.

  Among the companion animals, in the dog, the mammary tumor is the most common tumor and accounts for about half of the whole tumor in the female dog. Furthermore, half of the breast tumors eventually become malignant, ie breast cancer. Thus, breast cancer is a more dangerous disease for dogs than in other animals. Prolactin has been found to be a risk factor among factors involved in breast tumors. Furthermore, prolactin is desired to be measured in the behavioral field as a stress marker.

  At present, there are only a limited number of canine-specific hormone measurement systems, and thyroid-stimulating hormone (TSH) measurement system and growth hormone (GH) measurement system are available. Absent. As natural canine prolactin, there is a very small amount of research sample purified from a biological sample, but it is generally not provided and analysis of protein structure has not been made. Although the structure of the prolactin protein has been identified in several vertebrates, the variation between species is greater than that of other hormones, and the structure is unknown in dogs.

  Furthermore, since natural prolactin is contained in a very small amount in the pituitary gland, several hundred dogs are required for hormone extraction. However, it is very difficult to obtain it from the viewpoint of animal welfare. In addition, isolation and purification of prolactin from a biological sample itself is difficult, and the structure has not been analyzed.

  Development of an accurate measurement system for canine prolactin as a marker for breast cancer diagnosis and stress in dogs is desired, but obtaining prolactin in an amount necessary for measurement from a biological sample is a viewpoint of animal welfare. It was very difficult both economically and economically. Prolactin is species-specific, and it is necessary to produce a dog-specific antibody in a dog measurement system.

  Accordingly, an object of the present invention is to stably obtain an amount of canine prolactin necessary for accurately measuring canine prolactin.

  Therefore, the present inventors examined the cloning and determination of the canine prolactin gene in order to produce canine prolactin by gene recombination technology. To date, several putative sequences of canine prolactin have been reported in the Genbank database, but no studies have identified the sequence of the canine prolactin gene transcribed as mRNA.

  The present inventors have elucidated the structure of canine prolactin by cloning a canine prolactin gene from a canine pituitary cDNA library and expressing a recombinant protein, thereby completing the present invention.

That is, the present invention is as follows.
1. A canine prolactin gene having the base sequence represented by SEQ ID NO: 1.
2. A polynucleotide comprising the nucleotide sequence of positions 43 to 732 of SEQ ID NO: 1.
3. A recombinant canine prolactin precursor comprising a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
4). A polynucleotide comprising the nucleotide sequence of positions 133 to 732 of SEQ ID NO: 1.
5. A recombinant canine prolactin comprising a polypeptide consisting of the amino acid sequence at positions 31 to 229 of SEQ ID NO: 2.
6). A polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
7). A polynucleotide encoding a polypeptide consisting of the amino acid sequence of positions 31 to 229 of SEQ ID NO: 2.
8). A plasmid comprising the polynucleotide according to 6 above.
9. A plasmid comprising the polynucleotide according to 7 above.
10. 10. A cell transformed with the plasmid according to 8 or 9 above.
11. 10. A method for producing recombinant canine prolactin, which comprises culturing the cells described in 10 above to express canine prolactin and collecting the canine prolactin.

The present invention is described in detail below.
The canine prolactin gene was sequenced as follows. First, in order to construct a canine pituitary cDNA library, pituitary glands were collected from experimental animal dogs and RNA was extracted. This was reverse transcribed and converted to cDNA, and the cDNA was inserted into a plasmid. The gene sequence of each clone of this cDNA library was analyzed and searched in the database. Next, the base sequence of the full-length clone estimated to be prolactin was determined with reference to the canine genome database.

  The base sequence of the obtained clone is referred to as HM369390 (SEQ ID NO: 1). This base sequence is shown in FIG. 1 together with the deduced amino acid sequence, and the underlined portion indicates the deduced signal sequence. The double underlined part is the theoretical N-glycosylation site, and the asterisk (*) indicates the stop codon. The polyadenylation signal is indicated by a dashed line.

  Subsequently, the clone was introduced into HEK293 cells, and a stable expression cell line was selected. Canine prolactin was purified from the culture supernatant of HEK293 cells. For purification, gel filtration and reverse phase HPLC may be combined. The molecular weight was measured from the mobility of SDS-PAGE, and at the same time, the reactivity against the anti-prolactin antibody was confirmed by Western blot. In the analysis by Western blotting, the band detected by the anti-prolactin antibody was confirmed.

  The prolactin gene cloned in the present invention is judged to be canine prolactin from the fact that it matches the sequence deduced from the base sequence of the genome, the expressed protein reacts with the anti-prolactin antibody, and its molecular weight.

  The sequence HM369390 (SEQ ID NO: 1) identified in the present invention was different from the two sequences XM_545363 and AY741405 registered as canine prolactin genes in NCBI GenBank.

  BLAST (http://blast.ncbi.nlm.nih.gov/) analysis revealed that HM369390 is located on canine chromosome 35 and contains 5 exons and 4 introns. The sequence AY741405 was not detected on the canine chromosome. In addition, the sequence XM_545363 is identified in the same chromosome and contains 6 exons and 5 introns (FIG. 2), but this sequence is a sequence deduced from the genome sequence and confirmed to be actually transcribed into mRNA. It wasn't done. In contrast, the sequence HM369390 of the present invention is derived from mRNA sequenced from a canine pituitary cDNA library, and thus is considered to be a gene encoding an actual prolactin.

  Other types of prolactin have multiple splicing and / or translation variants, for example, the human prolactin gene consists of 6 exons, while the translated sequence consists of 5 exons. Rat prolactin has multiple biochemical variants due to post-translational modification of peptides, such as glycosylation, phosphorylation and cleavage.

  As shown in FIG. 2, XM_545363 shares exons II to V with HM369390, suggesting that HM369390 and XM_545363 can be splicing mutants, but XM_545363 is detected in the cDNA library prepared by the present inventors. Was not. For XM — 545363, it is considered that the start codon was set at a position different from the actual readout position when the deduced sequence was determined.

  As shown in FIG. 1, in the base sequence HM369390 (SEQ ID NO: 1), the 930 bp nucleotide sequence contains a complete open reading frame encoding a putative 229 amino acid polypeptide. The amino acid sequence of HM369390 contains 30 hydrophobic amino acids presumed to be signal peptides at its amino terminus and has one N-glycosylation site.

  The base sequence at positions 43 to 732 of SEQ ID NO: 1 represents the translation region, and the base sequence at positions 133 to 732 represents the region encoding mature prolactin excluding the region encoding the signal peptide. SEQ ID NO: 2 represents the amino acid sequence of the prolactin precursor containing the signal peptide, and corresponds to the nucleotide sequence at positions 43 to 732 of SEQ ID NO: 1. The amino acid sequence at positions 31 to 229 of SEQ ID NO: 2 represents the amino acid sequence of mature prolactin excluding the signal peptide.

  As described above, for the first time, the full length canine prolactin cDNA was isolated and sequenced according to the present invention. The deduced amino acid sequence was highly homologous to that of other species including cats and pigs. Furthermore, recombinant canine prolactin was expressed in mammalian cells and was found to be secreted as an N-linked glycosylated (31 kDa) or non-glycosylated (27 kDa) protein. The inventors have also found that the molecular weight of the recombinant protein is equivalent to that of natural prolactin extracted from the canine pituitary gland.

  Recombinant canine prolactin can be produced by using an expression vector containing a polynucleotide consisting of the nucleotide sequence of positions 43 to 732 of SEQ ID NO: 1 or the nucleotide sequence of positions 133 to 732 in mammalian cells or the like by a conventional method. It can be produced by introducing a cell into an appropriate host, selecting a cell in which a gene is integrated and stably expressing the transformed cell, and culturing the cell. Moreover, a prolactin recombinant can be stably produced by creating a stable expression strain.

  The recombinant canine prolactin prepared according to the present invention can be used for blood prolactin measurement for diagnosis and the like. Blood prolactin is usually measured by an immunoassay utilizing an antigen-antibody reaction. According to the present invention, a recombinant canine prolactin can be stably produced in large quantities, so that it is possible to produce a more specific antibody using this as an antigen. Prolactin has a large difference in animal species compared to other pituitary hormones, and canine prolactin cannot be accurately measured by a measurement system using prolactin of other animals.

  Examples of the immunoassay include time-resolved fluorescence immunoassay, and purified canine prolactin and anti-canine prolactin antibody, and europium-labeled canine prolactin are used for the measurement. In this case, not only can a specific antibody be produced using the recombinant prolactin obtained in the present invention as an antigen, but a recombinant prolactin can be used as a standard product (for a calibration curve) and a europium labeled hormone (tracer). That is, by using canine prolactin based on an accurate sequence as a standard for a calibration curve, an accurate measurement value can be obtained.

  Furthermore, the recombinant obtained by the present invention is useful in that a target for treatment of canine breast cancer can be studied by examining the protein function of canine prolactin.

  According to the present invention, the sequence of a gene transcribed as mRNA was accurately shown for the first time with respect to canine prolactin. If the polynucleotide encoding canine prolactin of the present invention is used, canine prolactin can be stably produced in a large amount using a gene recombination method. Further, the obtained recombinant canine prolactin can be used to produce a canine-specific anti-prolactin antibody, and a canine-specific prolactin measurement system that enables accurate measurement of this hormone in dogs can be established. . This measurement is expected to be useful as a marker in the diagnosis of prolactin-producing tumors and as a stress marker in the behavioral field.

FIG. 1 shows the nucleotide sequence of canine prolactin cDNA and the amino acid sequence found therefrom. FIG. 2 is a diagram showing the positions of the sequence HM369390 and the sequence XM_545363 on the canine chromosome 35. FIG. 3 shows the expression of recombinant canine prolactin prepared in Example 2 by Western blotting. FIG. 4 shows the results of treating recombinant canine prolactin with PNGase and analyzing it by Western blotting. FIG. 5 is a diagram showing a comparison of analysis results of recombinant canine prolactin and natural canine prolactin by Western blotting.

  The present invention will be described more specifically with reference to the following examples.

Identification of canine prolactin gene from canine pituitary cDNA library Pituitary gland was collected from adult beagle dogs, and total RNA was extracted using QIAGEN RNA preparation kit (RNAeasy). The production of cDNA clones from RNA was performed using a contract service of Hitachi High-Tech Manufacturing & Service Co., Ltd. 960 clones were obtained and sequenced.

  When the canine prolactin gene sequence was searched in the cDNA library and compared with the Genbank database, two clones having the same cDNA sequence (sequence HM369390, SEQ ID NO: 1) were detected. The sequence HM369390 was totally different, although it overlapped with the two sequences of registered canine prolactin genes, XM_545363 and AY741405.

  When the signal peptide of the deduced amino acid sequence of HM369390 is predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/), the 930 bp nucleotide sequence encodes a deduced 229 amino acid polypeptide. It included a fully open reading frame (Figure 1). The amino acid sequence of HM369390 contains 30 hydrophobic amino acids presumed to be signal peptides at its amino terminus and has one N-glycosylation site. The deduced amino acid sequence of HM369390 has 86.10% homology with XM — 545363 and 56.07% homology with AY741405. The gene for HM369390 also exhibits high amino acid homology with genes from other animal species, including cat and pig genes.

   By analyzing the sequence using BLAST (http://blast.ncbi.nlm.nih.gov/) and determining its genomic organization, HM369390 is located on canine chromosome 35 and contains 5 exons and 4 introns. I understood. The sequence AY741405 was not detected in the chromosome, but XM — 545363 was identified in the same chromosome and contains 6 exons and 5 introns (FIG. 2). The sequence XM_545363 is a sequence deduced from the genome sequence, and is not a sequence that has been confirmed to be actually transcribed into mRNA. XM_545363 shares exons II to V with HM369390, but XM_545363 was not detected in our cDNA library. In the sequence XM — 545363, it is considered that the start codon is set at a position different from the actual readout position when the estimated sequence is determined. In contrast, HM369390 sequenced from the canine pituitary cDNA library in the present invention is derived from mRNA and is considered to be an actual prolactin gene.

Preparation of recombinant canine prolactin To produce recombinant canine prolactin, a plasmid incorporating the prolactin gene was prepared. The full-length coding region of canine prolactin was amplified by PCR using cDNA (HM369390) as a template. PCR amplification with high fidelity polymerase (Expand HiFi: USA, IN, Indianapolis, Roche Diagnostics), 95 ° C, 15 sec denaturation, 60 ° C, 30 sec annealing, 72 ° C, 1 min polymerization, and 72 ° C A final polymerization of 10 minutes was performed under standard conditions of 30 cycles. As primers for canine prolactin, 5'-GCC GAA TTC ATC GCC ACC ATG GAT AAC AAA-3 '(SEQ ID NO: 3) and 5'-TAG GGA TCC TTA GCA GTT GCT GTC GTA GAC-3' (SEQ ID NO: 4) Was used. The PCR product was subcloned into the EcoRI and BamHI sites of pcDNA3.1 (-). The DNA sequence of the plasmid was confirmed by ABI PRISM 3730 Genetic Analyzer (Applied Biosystems, CA, Foster City) using Dye Deoxy Terminator Cycle Sequencing Kit. HM369390 expression vector (pcPRL) was transfected into human embryonic kidney (HEK) 293 cells (RIKEN Cell Bank, Tsukuba). These cells were maintained in DMEM / F12 (growth medium) (Invitrogen, CA, Carlsbad) supplemented with 10% heat-inactivated fetal bovine serum, 100 U / ml penicillin and 100 μg / mg streptomycin. Cells were seeded in 24-well plates at a density of 3 × 10 ⁵ cells / well and transfected with pcPRL using FuGene6 transfection reagent (Roche Diagnostics, IN, Indianapolis, USA). After 25 hours, the medium was replaced with serum-free medium (200 μL / well), and the cells were cultured for 4 days. The conditioned medium precipitated with acetone was treated with an anti-human prolactin antibody (Clone No. C-17: USA, CA, Santa Cruz Biotechnology) as described previously (Hashimoto, O. et al., 2006). Blot analysis was performed.

  As shown in FIG. 3, two signals that migrated to positions corresponding to 27 and 31 kDa were detected under reducing conditions. Recombinant canine prolactin is presumed to be glycosylated in HEK293 cells. Glycosylated prolactin has been reported to be an asparagine N-linked oligosaccharide in a single consensus sequence at positions 31-33 on the polypeptide chain. This glycosylation site has been identified in various species but has been identified in canine prolactin according to the present invention. To determine if the 31-kDa band of canine prolactin is an N-glycosylated form, conditioned media was treated with PNGase F (Nerengland Biolabs, USA, MA, Ipswich). Treatment with PNGase F disappeared the 31-kDa band and increased the strength of the 27-kDa band (FIG. 4). These results indicate that the 31-kDa band of canine prolactin is an N-glycosylated protein. Prolactin glycosylation is believed to alter its receptor binding capacity and / or its plasma half-life.

  Finally, conditioned media from cells transfected with pcPRL and prepared as previously reported (Hashimoto O. et al, 2006) to determine if recombinant canine prolactin protein and natural prolactin are equivalent. Western blotting using pituitary extracts from another female adult dog was performed. The signal band of the canine pituitary extract shifted to 27 and 31 kDa, as was the recombinant canine PRL protein (Figure 5). These results indicate that canine prolactin is secreted naturally as N-glycosylated (31 kDA) and non-glycosylated (27 kDa) prolactin, similar to the recombinant canine prolactin of the present invention.

Claims (11)

Canine prolactin gene having the nucleotide sequence represented by SEQ ID NO: 1.   A polynucleotide comprising the nucleotide sequence of positions 43 to 732 of SEQ ID NO: 1.   A recombinant canine prolactin precursor comprising a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.   A polynucleotide comprising the nucleotide sequence of positions 133 to 732 of SEQ ID NO: 1.   A recombinant canine prolactin comprising a polypeptide consisting of the amino acid sequence at positions 31 to 229 of SEQ ID NO: 2.   A polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.   A polynucleotide encoding a polypeptide consisting of the amino acid sequence of positions 31 to 229 of SEQ ID NO: 2.   A plasmid comprising the polynucleotide according to claim 6.   A plasmid comprising the polynucleotide according to claim 7.   A cell transformed with the plasmid according to claim 8 or 9.   A method for producing recombinant canine prolactin, comprising culturing the cells according to claim 10 to express canine prolactin and collecting the canine prolactin.