CN102676563A - Method for preparing human serum albumin-human parathyroid hormone - Google Patents

Abstract

The invention provides a method for preparing human serum albumin-human parathyroid hormone. By means of transforming a yeast host SMD1168, yapson1 coded sequences on the SMD 1168 can be knocked out. On the basis of the SMD 1168, coded gene YPS1 of yapson1 is further knocked out, namely double knockouts of a proteinase A and yapsin1 are achieved, and the transformed host is named as SMD1168yps1 delta. By the aid of the transformed host, a recombination expression of fusion proteins of human serum albumin-human parathyroid hormone (1-34) is optimized, so that the degradation of interest proteins in the expressing process can be reduced, and downstream mass production costs and difficulties of separation and purification are lowered. According to the method, the optimized the human serum albumin-human parathyroid hormone (1-34) fusion proteins can be applied to prepare parathyroid hormone (PTH) (1-34) medicines which are used for treating osteoporosis.

Description

A kind of method for preparing human serum albumin-human parathyroid hormone

Technical field

The invention belongs to biological technical field; Particularly; The structure that relates to pichia pastoris protein enzyme A and the yapsin 1 dual genetic engineering bacterium that knocks out, and this host is used for corresponding proteins lytic enzyme responsive type heterologous protein---human serum albumin-human parathyroid hormone (1-34) fusion rotein recombinant expressed.

Technical background

Pichia pastoris phaff (Pichia pastors) expression system is fitst water at present, one of most widely used exogenous protein expression system.It has not only overcome escherichia expression system can not complicated protein, the expressed proteins multiform of expression structure become defectives such as insoluble inclusion body, background albumen is many, expression yields poorly; Remedy cells of mamma animals, insect cell expression system complicated operation, expression level is low, industrialization production involves great expense deficiency, also had an incomparable superior part of other yeast expression systems.The advantage of this expression system mainly contain following some: (1) has strong alcohol oxidase promotor (P _AOX1), expression that can strict regulation and control foreign protein; (2) high-density large-scale that is easy to be implemented in the synthetic medium is cultivated, pichia spp self be secreted in the substratum albumen seldom, high excretory foreign protein is prone to from protein free substratum, separate.(3) degree of glycosylation is low, compares with yeast saccharomyces cerevisiae, and pichia spp does not produce the over-drastic glycosylation; The immunogenicity of secreted gp is lower, is more conducive to clinical application.Utilize at present this expression system successful expression nearly 500 kinds of heterologous proteins.

Yet; The degraded of recombinant expressed foreign protein; The proteolytic degradation that particularly causes by the multiple protein lytic enzyme that exists in the pichia spp born of the same parents; Usually make the expression amount of foreign protein reduce greatly, because the physico-chemical property of degraded product and intact proteins is very close, also the separation and purification in downstream is had higher requirement simultaneously.This also is the bottleneck problem of pichia yeast expression system development.When the optimization culture condition can't reach the purpose that reduces the foreign protein degraded; Usually can consider to utilize genetic engineering means that active proteolytic ferment is knocked out; As the SMD1168 of the use of commercialization, the PEP4 gene of proteins encoded enzyme A is knocked out in this strain gene group, causes the protease A loss of activity; Also partly reduce the activity of proteolytic enzyme B and carboxypeptidase y simultaneously, made expression product avoid the degraded of this proteinoid enzyme.

Yet host SMD1168 can only be effective to some specific albumen, and the degraded that not all exogenous protein expression produces can both be effectively suppressed.Early stage research is divided into three major types with zymic intracellular protein enzyme: the proteolytic enzyme of vacuole protein enzyme, cell matrix proteasome, Secretory Pathway according to the secretion and the where-used of proteolytic enzyme.Yapsins (the respective coding gene is called YPS) is newfound in recent years one type of proteolytic ferment that is present on the yeast emiocytosis approach; Belong to aspartic acid proteolyze enzyme family; It is characterized in that having one section glycosylation PI structure (GPI), it is anchored on the cytolemma.This proteinoid lytic enzyme is the enzymolysis site with single on the substrate or paired basic aminoacids sequence mainly.First identified out and study more deep be the yapsin 1 in the yeast saccharomyces cerevisiae.In other fungies, also identified successively afterwards and had multiple yapsin member.Yapsin 1 in the pichia spp identifies in 2005 by Wertem etc. first, and regrettably, they find that GS115 bacterial strain that YPS1 knocks out does not improve effect for the degraded of gelatin.But more and more evidences shows that the yapsins defective has significant improvement effect to the degraded of some specific protein in yeast saccharomyces cerevisiae or the lactic acid Crewe dimension genus yeast.As the yeast saccharomyces cerevisiae of using yapsin 1 defective can reduce the degraded of reorganization PTH greatly.People such as Schutter had announced pichia spp gene order-checking result in 2009, through sequential analysis, find except YPS1, in the pichia spp born of the same parents, also to have 6 YPS members, and be respectively YPS2, YPS2 ", YPS3, YPS3 " and, YPS7 and MKC7.

Summary of the invention

The purpose of this invention is to provide a kind of method for preparing human serum albumin-human parathyroid hormone.At first, the yeast host SMD1168 that is bought by Invitrogen is transformed,, knock out the encoding sequence (SEQ ID NO.11) of yapsin 1 on the SMD1168 genome promptly through the method for homologous recombination.SMD1168 is that a kind of protease A knocks out the type host, and it is the gene knockout gained with proteins encoded enzyme A among the host GS115.The present invention further knocks out the encoding sox YPS1 of yapsin 1 on the basis of SMD1168 and has realized that promptly protease A and the dual of yapsin 1 knock out, and should transform host's called after SMD1168yps1 Δ.Utilize above-mentioned transformation host to optimize recombinant expressed human serum albumin-human parathyroid hormone (1-34) fusion rotein again, thereby reduce the degraded of the target protein in the expression process.

The present invention realizes through following steps: (a) 5 ' the end homology arm of design YPS1 and 3 ' end homology arm amplimer, and from the pichia spp genome, effectively amplify this two homology arm sequences; (b) these two homology arm sequences that obtain in the step (a) are inserted among the carrier pPICZ α B according to sequencing, obtain to be used for the recombinant vectors pPICZ α B-YPS1 Δ of subsequent homo reorganization; (c) with the recombinant vectors that makes up in the step (b) with single digestion with restriction enzyme after transfection SMD1168; Cultivation screening recon with the selective pressure that contains Zeocin; With the exactness of corresponding positive primer and negative primer checking homologous recombination, correct defective type recon is designated as the SMD1168yps1 Δ; (d) utilize the protease-deficient host SMD1168yps1 Δ that obtains in the step (c) to express that corresponding proteins lytic enzyme responsive type recombinant protein---human serum albumin-human parathyroid hormone (1-34) fusion rotein, SEQ ID NO:13 are the gene and the protein sequence of this fusion rotein.

In the step (a); The invention provides the primer sequence that is used for YPS15 ' end homology arm and 3 ' end homology arm amplification; The gene order (SEQ ID NO.9) of YPS15 ' end homology arm is provided simultaneously, the gene order (SEQ ID NO.10) of YPS13 ' end homology arm and coding yapsin 1 gene order YPS1 (SEQ ID NO.11).Wherein, be used to the to increase upstream primer of YPS15 ' end homology arm sequence is: SEQ ID NO.1:5 '-aagc CtgcagAgctccattg cgccaacccc-3 ', the line part is a Pst I restriction enzyme site, downstream primer is: SEQ ID NO.2:5 '-cggc GtcgacAatctggctg agcggaaagt ttga-3 ', the line part is the SalI restriction enzyme site.The YPS13 ' that is used to increase holds the upstream primer of homology arm sequence to be: SEQ ID NO.3:5 '-ccc AgatctC acatttcgcg cctgccttcc t-3 ', the line part is the BglII restriction enzyme site, downstream primer is: SEQ ID NO.4:5 '-aaa CtgcagG ggtgagcctg tctggccct-3 ', the line part is a Pst I restriction enzyme site.

In the step (b); The invention provides the recombinant vectors that carries YPS15 ' end homology arm and 3 ' end homology arm dna sequence dna; Confirm that through PCR checking and dna sequencing above-mentioned two homology arms are inserted among the carrier pPICZ α B with correct mode, are designated as pPICZ α B-YPS1 Δ (SEQ ID NO.12) with recombinant vectors.

In the step (c); The present invention is according to the change that sequence takes place before and after homologous recombination of zymic target homologous recombination zone; Design a pair of negative primer (SEQ ID NO.5:5 '-ctccttccac cgccacggc-3 '; SEQ ID NO.6:5 '-accacttgag gcgtccttgg a-3 ') confirms that the encoding sequence of YPS1 in the recon is knocked out; Further a pair of positive primer of design (SEQ ID NO.7:5 '-atggaggagt tgaggacc-3 ', SEQ ID NO.8:5 '-gcaaatggca ttctgacatc c-3 ') is proved conclusively the exactness of homologous recombination position again.

Utilize the known method in this area can accomplish the structure of homologous recombination vector, the extraction of yeast genes group can be with reference to (A. Adam Si etc., " yeast genetics method experiment guide ", Science Press, 2000).The acquisition of YPS15 ' end homology arm and 3 ' end homology arm can be used the method for PCR Science.239:487-491 such as (, 1988) Saiki or synthetic.Like method through PCR, introduce the restriction enzyme enzyme recognition site in the both sides of target sequence, cut the generation sticky end through enzyme, can be cloned into required carrier easily.The molecular cloning process of used standard is seen (J. Sa nurse Brooker etc., " molecular cloning experiment guide " second edition, Science Press, 1995.).

Yeast host GS115, SMD1168 and expression vector pPICZ α B can buy (Invitrogen Corp.San Diego.California.USA) from company.Preferable methods is that 5 ' end and 3 ' end homology arm are cloned into carrier pPICZ α B.The Zeocin resistant gene that this carrier carries can be used as the colony screening mark after the subsequent homo reorganization.

Transform and go the available common method that gets in required nucleic acid to the host cell, like electroporation, the competent spheroplast of preparation etc.The success cell transformed can be identified through the technology that people know, and through collecting and cracking, extracts DNA like cell, and PCR method is identified then.Specifically can be with reference to implementing regulations 3.

Human serum albumin-human parathyroid hormone (1-34) fusion rotein (HSA/PTH (1-34)) utilizes genetic engineering means that both genes are merged; Last a kind of long-acting human parathyroid hormone (1-34) form of in pichia spp, expressing acquisition; This albumen is a kind of very promising pharmaceutical protein that can treat osteoporosis, sees granted patent (application number 200410066459.8).The applicant finds when adopting pichia spp GS115 as this proteic expressive host in earlier stage; Produce human serum albumin-human parathyroid hormone fusion rotein though utilize GS115 to express with BA; But there is serious signs of degradation in expression product; Cause the expression amount of intact proteins not high, and will pass through more purification procedures and just can obtain highly purified intact proteins, the productive rate of recombinant protein is extremely low.Use commercial defective type host SMD1168 also can only partly suppress the to degrade generation of band instead, prompting also has other proteolytic ferments in the recombinant expressed process of HSA/PTH (1-34), to play a leading role except protease A.This experiment of later stage is through making up 7 kinds of single defective hosts that knock out of YPS, i.e. GS115yps1 Δ, GS115yps2 Δ; GS115yps2 " Δ, GS115yps3 Δ, GS115yps3 " Δ; The GS115yps7 Δ; The GS115mkc7 Δ, only in the host that YPS1 knocks out, the degraded of HSA/PTH (1-34) obviously reduces in discovery.According to this result, we have confirmed that yapsin 1 is another principal element that causes HSA/PTH (1-34) degraded.A kind of method that makes up protease A and the yapsin 1 dual protease-deficient pichia spp host who knocks out is provided; Utilize protease A and the yapsin 1 dual pichia spp that knocks out as expressive host; And utilize this host to optimize the recombinant expressed of human serum albumin-Rat parathyroid hormone 1-34 (1-34) fusion rotein; Reduce the signs of degradation of human serum albumin-human parathyroid hormone (1-34) fusion rotein in the expression process, thereby reduce downstream scale operation cost and separation and purification difficulty, when improving expression amount, also help separation and purification; Make this albumen when industrial amplification production, reduce difficulty, reduce cost.Lay the foundation for further studying treatment osteoporotic PTH (1-34) long lasting drug formulations.

Figure of description

Fig. 1 is the structure synoptic diagram of recombinant plasmid pPICZ α B-YPS1 Δ.

Fig. 2 is the PCR proof diagram of recombinant plasmid pPICZ α B-YPS1 Δ.

Fig. 3 identifies synoptic diagram for structure synoptic diagram and the PCR that homologous recombination method knocks out the YPS1 gene.

Fig. 4 is that the PCR of host SMD1168yps1 Δ identifies electrophorogram.

The PAGE that Fig. 5 expresses in normal host GS115 and protease-deficient host SMD1168 and SMD1168yps1 Δ for fusion rotein analyzes.

Embodiment

The present invention combines accompanying drawing and specific embodiment to do further explanation.

The amplification of embodiment 1:YPS15 ' end homology arm and 3 ' end homology arm

5 ' the end homology arm fragment and 3 ' that from the yeast genes group, obtains the YPS1 sequence with PCR method is held the homology arm fragment, and used primer YPS1_N up (SEQ ID NO.1), YPS1_N dn (SEQ ID NO.2) and YPS1_C up (SEQ ID NO.3), YPS1_C dn (SEQ ID NO.4) synthesize with the oligonucleotide synthesizer.The upstream and downstream primer that is used for 5 ' end homology arm amplification is introduced PstI and Sa1I site and protection base respectively, and the upstream and downstream primer that is used for 3 ' end homology arm amplification is introduced BglII and PstI site and protection base respectively, and line place is the restriction endonuclease recognition sequence.

YPS1_N up：5’-aagc ctgcag agctccattg cgccaacccc-3’

YPS1_N dn：5’-cggc gtcgac aatctggctg agcggaaagt ttga-3’

YPS1_C up：5’-ccc agatctc acatttcgcg cctgccttcc t-3’

YPS1_C dn：5’-aaa ctgcagg ggtgagcctg tctggccct-3’

PCR reaction conditions: in the 50 μ L reaction systems, add 1 μ L yeast genes group, each 1 μ L of the upstream and downstream primer of 10 μ mol/L, the dNTP mixture 4 μ L of 2.5mmol/L, 5 * Buffer (Mg ²⁺Plus) 10 μ L, PrimeSTAR archaeal dna polymerase 0.5 μ L, residue is used ddH ₂O supplies.With (model is Matstercycler Gradient) PCR appearance of EPPENDORF company, the PCR reaction conditions is 98 ℃ of preparatory sex change 5 minutes; 98 ℃ of sex change 10s; 55 ℃ of annealing 15min; 72 ℃ are extended 30s, and after totally 30 circulations, 72 ℃ are extended 10min again.Obtain being contemplated to 5 ' the end homology arm sequence of 257bp and 3 ' the end homology arm sequence of 318bp through 1.5% gel electrophoresis analysis.

Embodiment 2: the structure of recombinant vectors pPICZ α B-YPS1 Δ

Reclaim purifying purpose band with reclaiming test kit with dna fragmentation behind 3 ' the end homology arm PCR product electrophoresis purifying that obtains among the embodiment 1.Behind the purifying 3 ' end homology arm PCR product is cut with the BglII/PstI enzyme, and electrophoresis reclaims; Plasmid pPICZ α B cuts with the BglII/PstI enzyme equally, and electrophoresis reclaims the linearizing fragment.3 ' end homology arm after enzyme cut mixes with suitable proportion with carrier pPICZ α B, connects in 16 ℃ of water-baths with the T4 ligase enzyme and spends the night, formation pPICZ α B-C.After being transformed into DH5 α, be laid on the LB agar plate that contains 25 μ g/mL Zeocin, 37 ℃ of overnight cultures.Select on the plate several clone's inoculations 5mL and contain in the LB liquid nutrient medium of 25 μ g/mL Zeocin 37 ℃ of overnight cultures.With primer YPS1_C up/YPS1_C dn is carried out the PCR checking and obtain positive colony pPICZ α B-C/DH5 α (see figure 2), it is correct that positive colony is served the further sequence verification of Hai Shenggong.

Reclaim purifying purpose band with reclaiming test kit with dna fragmentation behind 5 ' the end homology arm PCR product electrophoresis purifying that obtains among the embodiment 1.Behind the purifying 5 ' end homology arm PCR product is cut with the PstI/SalI enzyme, and electrophoresis reclaims; Plasmid pPICZ α B-C cuts with the PstI/SalI enzyme equally, and electrophoresis reclaims the linearizing fragment.5 ' end homology arm after enzyme cut mixes with suitable proportion with carrier pPICZ α B-C, connects in 16 ℃ of water-baths with the T4 ligase enzyme and spends the night, formation pPICZ α B-YPS1 Δ.After being transformed into DH5 α, be laid on the LB agar plate that contains 25 μ g/mL Zeocin, 37 ℃ of overnight cultures.Select on the plate several clone's inoculations 5mL and contain in the LB liquid nutrient medium of 25 μ g/mLZeocin 37 ℃ of overnight cultures.With primer YPS1_C up/YPS1_N dn is carried out the PCR checking and obtain positive colony pPICZ α B-YPS1 Δ/DH5 α (see figure 2), it is correct that positive colony is served the further sequence verification of Hai Shenggong.

Embodiment 3: homologous recombination method knocks out the YPS1 gene among the host SMD1168

Therefore pichia spp host SMD1168 is protease A defective type host, on the basis of SMD1168, aspartic acid lytic enzyme 1 gene is further knocked out and can obtain protease A and the yapsin 1 dual pichia spp host who knocks out.

The mode that pPICZ α B-YPS1 Δ is transformed with electricity after with the PstI linearizing is transformed into pichia spp SMD1168 (the yeast expression test kit is provided by Invitrogen Corp.USA); Hatching 2h in the YPD liquid nutrient medium after, SMD1168 after the conversion is laid on YPD Agr plate (the 1%yeast extract that contains 50 μ g/mL Zeocin and 1M sorbyl alcohol; 2%peptone, 2% glucose, 1M sorbyl alcohol; 2% agarose) on, cultivated 3-5 days for 30 ℃.Several clones are inoculated in 5mL and contain in the YPD liquid nutrient medium of 50 μ g/mL Zeocin 30 ℃ of overnight cultures on the picking plate.PCR verifies positive colony; Concrete mode is seen Fig. 3, Fig. 4: design a pair of negative primer (yps1 negative up (SEQ ID NO.5), yps1 negative dn (SEQ ID NO.6)), a pair of positive primer (yps1 positive up (SEQ ID NO.7); Yps1 positive dn (SEQ ID NO.8)); The positive colony that the correct YPS1 that recombinates knocks out is designated as the SMD1168yps1 Δ, can amplify the 833bp band with positive primer, can not amplify any band with negative primer.

yps1 negative up：5’-ctccttccac cgccacggc-3’

yps1 negative dn：5’-accacttgag gcgtccttgg a-3’

yps1 positive up：5’-atggaggagt tgaggacc-3’

yps1 positive dn：5’-gcaaatggca ttctgacatc c-3’

Embodiment 4: the expression of fusion rotein HSA/PTH (1-34) in host GS115

Fusion rotein HSA/PTH (1-34) (SEQ ID NO.13) applies for a patent and authorizes (application number 200410066459.8) by this laboratory.Described in this patent 200410066459.8, the mode that recombinant expression vector pPIC9-HSA/PTH (1-34) is transformed with electricity after with Sal I linearizing is transformed into pichia spp GS115, and detailed electric transform mode can be with reference to Invitrogen yeast laboratory manual.Conversion fluid is laid on RDB agar plate (1mol/L sorbyl alcohol, 1% glucose, 4*10 ^-5The % vitamin H, 1.34% no amino acid yeast nitrogen (YNB, Difeocorp.), 0.005% amino acid mixing liquid (L-glutamic acid, methionine(Met), Methionin, leucine, Isoleucine each 0.005%), 2% agarose) on, 3～5d, picking His cultivated for 30 ℃ ⁺The clone is seeded to 20mL BMGY substratum (1%yeast extract, 2%peptone, 100mmol/L potassiumphosphate, pH6.0,4*10 is housed ^-5% vitamin H, 1.34% no amino acid yeast nitrogen, 1% glycerine) the 100mL triangular flask, 250 rev/mins; 30 ℃ be cultured to OD=2～6 after, change 20mL BMMY substratum (1%yeast extract, 2%peptone into; The 100mmol/L potassiumphosphate, pH6.0,4*10 ^-5% vitamin H, 1.34% no amino acid yeast nitrogen, 1% methyl alcohol), after this add 1% methyl alcohol/24h, induce centrifuging and taking supernatant behind the 3d, native-PAGE analyzes expression product, and the result finds that referring to Fig. 5 there is serious degraded in the fusion rotein in the culture supernatant.

Embodiment 5: the expression of fusion rotein HSA/PTH (1-34) in host SMD1168

Described in patent No. zl200410066459.8; The mode that recombinant expression vector pPIC9-HSA/PTH (1-34) is transformed with electricity after with Sal I linearizing is transformed into pichia spp SMD1168, and detailed electric transform mode can be with reference to Invitrogen yeast laboratory manual.Conversion fluid is laid on RDB agar plate (1mol/L sorbyl alcohol, 1% glucose, 4*10 ^-5The % vitamin H, 1.34% no amino acid yeast nitrogen (YNB, Difeocorp.), 0.005% amino acid mixing liquid (L-glutamic acid, methionine(Met), Methionin, leucine, Isoleucine each 0.005%), 2% agarose) on, 3～5d, picking His cultivated for 30 ℃ ⁺The clone is seeded to 20mL BMGY substratum (1%yeast extract, 2%peptone, 100mmol/L potassiumphosphate, pH6.0,4*10 is housed ^-5% vitamin H, 1.34% no amino acid yeast nitrogen, 1% glycerine) the 100mL triangular flask, 250 rev/mins; 30 ℃ be cultured to OD=2～6 after, change 20mL BMMY substratum (1%yeast extract, 2%peptone into; The 100mmol/L potassiumphosphate, pH6.0,4*10 ^-5The % vitamin H, 1.34% no amino acid yeast nitrogen, 1% methyl alcohol); After this add 1% methyl alcohol/24h, induce centrifuging and taking supernatant behind the 3d, native-PAGE analyzes expression product; The result is referring to Fig. 5; Though find that the degraded of the target protein that host SMD1168 is produced is alleviated to some extent with respect to host GS115, the percentage composition of degraded band in culture supernatant is still greater than 50% in SMD1168.

The optimization expression of embodiment: 6: fusion rotein HSA/PTH (1-34) in host SMD1168yps1 Δ of the present invention

The mode that recombinant expression vector pPIC9-HSA/PTH (1-34) described in the patent No. z1200410066459.8 is transformed with electricity after with Sal I linearizing is transformed into pichia spp SMD1168 yps1 Δ, and detailed electric transform mode can be with reference to Invitrogen yeast laboratory manual.Conversion fluid is laid on RDB agar plate (1mol/L sorbyl alcohol, 1% glucose, 4*10 ^-5The % vitamin H, 1.34% no amino acid yeast nitrogen (YNB, Difeocorp.), 0.005% amino acid mixing liquid (L-glutamic acid, methionine(Met), Methionin, leucine, Isoleucine each 0.005%), 2% agarose) on, 3～5d, picking His cultivated for 30 ℃ ⁺The clone is seeded to 20mL BMGY substratum (1%yeast extract, 2%peptone, 100mmol/L potassiumphosphate, pH6.0,4*10 is housed ^-5% vitamin H, 1.34% no amino acid yeast nitrogen, 1% glycerine) the 100mL triangular flask, 250 rev/mins, 30 ℃ be cultured to OD=2～6 after, change 20mL BMMY substratum (1%yeastextract, 2%peptone, 100mmol/L potassiumphosphate, pH6.0,4*10 into ^-5% vitamin H, 1.34% no amino acid yeast nitrogen, 1% methyl alcohol), after this add 1% methyl alcohol/24h, induce centrifuging and taking supernatant behind the 3d, native-PAGE analyzes expression product.The result is referring to Fig. 5, and than host GS115 and SMD1168, in host SMD1168 yps1 Δ, the degraded band has obtained maximum inhibition, explains that the SMD1168yps1 Δ is optimum relatively a kind of host.

< 110>Zhejiang University

< 120>a kind of method for preparing human serum albumin-human parathyroid hormone

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cccagatctc acatttcgcg cctgccttcc t 31

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accacttgag gcgtccttgg a 21

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< 223>be used to identify the positive downstream primer of SMD1168 yps1 △ recon

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gcaaatggca ttctgacatc c 21

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< 223>pichia spp GS115 YPS1 5' homology arm gene order

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agctccattg cgccaacccc cgctctccag actccttcgt tatccagcat tcagcatgga 60

caggttcaaa aaataaaatt tcttgatatg ggtccacttc aaacatgcgc ctacctgtag 120

gaaaaaaaaa gagaacataa atatgccgcg aacagaaaac gtaatgtact gttctatata 180

taaactgttc agatcaatca taaattctca gtttcaaact ttccgctcag ccagatt 237

<210> 10

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<213> Pichia pastoris

<220>

< 223>pichia spp GS115 YPS1 3' homology arm gene order

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cacatttcgc gcctgccttc ctttaggttc tttgaatcat catcaatcgt cgccgtctac 60

atcagagcag gacttatctt tgccttcccc aaaaattgcc actccgtcaa atagattctt 120

ttgaatcctt gactattttt gcctaaatag gtttttgtta gtttttcttc aaagcccaaa 180

agaaactcta tttagattca tccagaaaca atctttttct caccccattt cgaagtgccg 240

tggagcacag acataaaaag atgactaccg ttcaacctac agggccagac aggctcaccc 300

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<211> 1800

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<213> Pichia pastoris

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<221> CDS

<222> (1)~(1797)

< 223>pichia spp GS115 YPS1 sequence

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gt ttt tag ctc cat tgc gcc aac ccc cgc tct -240

cca gac tcc ttc gtt atc cag cat tca gca tgg aca ggt tca aaa aat aaa att tct tga -180

tat ggg tcc act tca aac atg cgc cta cct gta gga aaa aaa aag aga aca taa ata tgc -120

cgc gaa cag aaa acg taa tgt act gtt cta tat ata aac tgt tca gat caa tca taa att -60

ctc agt ttc aaa ctt tcc gct cag cca gat ttt att cgt aaa gaa cgc atc att ggc tct 0

atg ttg aag gat cag ttc ttg tta tgg gtt gct ttg ata gcg agc gta ccg gtt tcc ggc 60

Met Leu Lys Asp Gln Phe Leu Leu Trp Val Ala Leu Ile Ala Ser Val Pro Val Ser Gly

1 5 10 15 20

gtg atg gca gct cct agc gag tcc ggg cat aac acg gtt gaa aaa cga gat gcc aaa aac 120

Val Met Ala Ala Pro Ser Glu Ser Gly His Asn Thr Val Glu Lys Gln Asp Ala Lys Asn

21 25 30 35 40

gtt gtt ggc gtt cga cag ttg gac ttc agc gtt ctg agg ggt gat tcc ttc gaa agt gcc 180

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

41 45 50 55 60

tct tca gag aac gtg cct cgg ctt gtg agg aga gat gac acg cta gaa gct gag cta atc 240

Ser Ser Glu Asn Val Pro Arg Leu Val Arg arg Asp Asp Thr Leu Glu Ala Glu Leu Ile

61 65 70 75 80

aac cag caa tca ttc tac ttg tca cga ctg aaa gtt gga tca cat caa gcg gat att gga 300

Asn Gln Gln Ser Phe Tyr Leu Ser Arg Leu Lys Val Gly Ser His Gln Ala Asp Ile Gly

81 85 90 95 100

atc cta gtg gac aca gga tcc tct gat tta tgg gta atg gac tcg gta aac cca tac tgc 360

Ile Leu Val Asp Thr Gly Ser Ser Asp Leu Trp Val Met Asp Ser Val Asn Pro Tyr Cys

101 105 110 115 120

agt agc cgt tcc cgc gtg aag aga gat ata cac gat gag aag atc gcc gaa tgg gat ccc 420

Ser Ser Arg Ser Arg Val Lys Arg Asp Ile His Asp Glu Lys Ile Ala Glu Trp Asp Pro

121 125 130 135 140

atc aat ctc aag aaa aat gaa act tct cag aat aaa aat ttt tgg gat tgg ctc gtt gga 480

Ile Asn Leu Lys Lys Asn Glu Thr Ser Gln Asn Lys Asn Phe Trp Asp Trp Leu Val Gly

141 145 150 155 160

act agc act agt tct cct tcc acc gcc acg gca act ggt agt ggt agt ggt agt ggt agt 540

Thr Ser Thr Ser Ser Pro Ser Thr Ala Thr Ala Thr Gly Ser Gly Ser Gly Ser Gly Ser

161 165 170 175 180

ggt agt ggt agt ggt agt gct gcc aca gcc gta tcg gta agt tct gca cag gca aca ttg 600

Gly Ser Gly Ser Gly Ser Ala Ala Thr Ala Val Ser Val Ser Ser Ala Gln Ala Thr Leu

181 185 190 195 200

gat tgc tct acg tat gga acg ttt gat cac gct gat tcc tcg acg ttc cat gac aat aat 660

Asp Cys Ser Thr Tyr Gly Thr Phe Asp His Ala Asp Ser Ser Thr Phe His Asp Asn Asn

201 205 210 215 220

aca gac ttt ttc atc tca tac gct gat acc act ttt gct tca gga atc tgg ggt tat gac 720

Thr Asp Phe Phe Ile Ser Tyr Ala Asp Thr Thr Phe Ala Ser Gly Ile Trp Gly Tyr Asp

221 225 230 235 240

gac gtc att atc gac ggc ata gag gtg aaa gaa ctt tcc ttc gcc gtt gca gac atg acc 780

Asp Val Ile Ile Asp Gly Ile Glu Val Lys Glu Leu Ser Phe Ala Val Ala Asp Met Thr

241 245 250 255 260

aat tcc tct att ggt gtg tta ggt att gga ctg aaa ggc cta gaa tcc aca tat gct agt 840

Asn Ser Ser Ile Gly Val Leu Gly Ile Gly Leu Lys Gly Leu Glu Ser Thr Tyr Ala Ser

261 265 270 275 280

gca tct tcg gtc agt gaa atg tat cag tat gac aat ttg cca gcc aag atg gtc acc gat 900

Ala Ser Ser Val Ser Glu Met Tyr Gln Tyr Asp Asn Leu Pro Ala Lys Met Val Thr Asp

281 285 290 295 300

ggg ttg atc aac aaa aat gca tac tcc ttg tac ttg aac tcc aag gac gcc tca agt ggt 960

Gly Leu Ile Asn Lys Asn Ala Tyr Ser Leu Tyr Leu Asn Ser Lys Asp Ala Ser Ser Gly

301 305 310 315 320

tcc atc ctc ttt gga ggt gtg gat cat gaa aaa tat tcg gga caa ttg ttg aca gtt cca 1020

Ser Ile Leu Phe Gly Gly Val Asp His Glu Lys Tyr Ser Gly Gln Leu Leu Thr Val Pro

321 325 330 335 340

gtc atc aac aca ctc gct tcc agt ggt tac aga gag gca att cgt tta caa att act tta 1080

Val Ile Asn Thr Leu Ala Ser Ser Gly Tyr Arg Glu Ala Ile Arg Leu Gln Ile Thr Leu

341 345 350 355 360

aat gga ata gat gtg aaa aag ggt tct gac cag gga act ctt tta caa ggg aga ttt gct 1140

Asn Gly Ile Asp Val Lys Lys Gly Ser Asp Gln Gly Thr Leu Leu Gln Gly Arg Phe Ala

361 365 370 375 380

gca tta ttg gac tct gga gct acg cta acg tat gct cct tct tct gtt tta aat tca att 1200

Ala Leu Leu Asp Ser Gly Ala Thr Leu Thr Tyr Ala Pro Ser Ser Val Leu Asn Ser Ile

381 385 390 395 400

ggc cgg aac ctg ggc ggc tcc tat gat tcg tca aga caa gct tat acc att cgt tgt gtt 1260

Gly Arg Asn Leu Gly Gly Ser Tyr Asp Ser Ser Arg Gln Ala Tyr Thr Ile Arg Cys Val

402 405 410 415 420

tct gca tca gat acc act tct ctg gta ttc aat ttt ggg ggt gct aca gtg gaa gtt tcc 1320

Ser Ala Ser Asp Thr Thr Ser Leu Val Phe Asn Phe Gly Gly Ala Thr Val Glu Val Ser

421 425 430 435 440

ctg tac gat cta cag att gca aca tat tac acc ggg gga agt gcc acg caa tgt ctt att 1380

Leu Tyr Asp Leu Gln Ile Ala Thr Tyr Tyr Thr Gly Gly Ser Ala Thr Gln Cys Leu Ile 460

441 445 450 455 460

gga ata ttc agc tct gga agt gat gag ttt gtg ctc ggt gat acc ttc ttg agg tca gcc 1440

Gly Ile Phe Ser Ser Gly Ser Asp Glu Phe Val Leu Gly Asp Thr Phe Leu Arg Ser Ala 480

461 465 470 475 480

tac gtg gtt tac gat ctt gat ggg ctt gaa gtg tcg ctt gcc caa gcc aac ttc aac gaa 1500

Tyr Val Val Tyr Asp Leu Asp Gly Leu Glu Val Ser Leu Ala Gln Ala Asn Phe Asn Glu 500

481 485 490 495 500

acc gat tct gat gtt gag gct att acc tcc agt gta cct tcc gct act cgt gca tcc gga 1560

Thr Asp Ser Asp Val Glu Ala Ile Thr Ser Ser Val Pro Ser Ala Thr Arg Ala Ser Gly 520

501 505 510 515 520

tac agt tct aca tgg tct ggt tct gcc agc ggt aca gtt tac act tcg gtt cag atg gaa 1620

Tyr Ser Ser Thr Trp Ser Gly Ser Ala Ser Gly Thr Val Tyr Thr Ser Val Gln Met Glu 540

521 525 530 535 540

tcc ggt gct gct tcc agc tcc aac tct tct gga tcg aat atg ggt tcc tct tcc tca tcg 1680

Ser Gly Ala Ala Ser Ser Ser Asn Ser Ser Gly Ser Asn Met Gly Ser Ser Ser Ser Ser 560

541 545 550 555 560

tcc tct tca tcg tcc tcg act tcc agt gga gac gaa gaa gga ggg agc tcc gcc aac agg 1740

Ser Ser Ser Ser Ser Ser Thr Ser Ser Gly Asp Glu Glu Gly Gly Ser Ser Ala Asn Arg 580

561 565 570 575 580

gtc ccc ttc agc tac ctt tct ctc tgt ttg gta gtt att ctc ggc gtg tgt ata gta tag 1800

Val Pro Phe Ser Tyr Leu Ser Leu Cys Leu Val Val Ile Leu Gly Val Cys Ile Val 599

581 585 590 595

tat aaa agg gcc tac att gga tag gct tca aca ttc ctc aat aaa caa aca tcc aac atc 1860

gcg cat tcc gca ttt cgc att tca cat ttc gcg cct gcc ttc ctt tag gtt ctt tga atc 1920

atc atc aat cgt cgc cgt cta cat cag agc agg act tat ctt tgc ctt ccc caa aaa ttg 1980

cca ctc cgt caa ata gat tct ttt gaa tcc ttg act att ttt gcc taa ata ggt ttt tgt 2040

tag ttt ttc ttc aaa gcc caa aag aaa ctc tat tta gat tca tcc aga aac aat ctt ttt 2100

ctc acc cca ttt cga agt gcc gtg gag cac aga cat aaa aag atg act acc gtt caa cct 2160

aca ggg cca gac agg ctc acc ctg ccg cat att cta ctg gaa ttc aac gat ggc tcc tcg 2220

cag cat gca gtg atc gag cta agc atg aac gag ggg att aat ata tcc acc cat gag tgg 2280

<210> 12

<211> 2829

<212> DNA

< 213>artificial sequence

<220>

< 223>dna sequence dna of recombinant vectors pPICZ α B-YPS1 △

<440> 12

agatctcaca tttcgcgcct gccttccttt aggttctttg aatcatcatc aatcgtcgcc 60

gtctacatca gagcaggact tatctttgcc ttccccaaaa attgccactc cgtcaaatag 120

attcttttga atccttgact atttttgcct aaataggttt ttgttagttt ttcttcaaag 180

cccaaaagaa actctattta gattcatcca gaaacaatct ttttctcacc ccatttcgaa 240

gtgccgtgga gcacagacat aaaaagatga ctaccgttca acctacaggg ccagacaggc 300

tcacccctgc agagctccat tgcgccaacc cccgctctcc agactccttc gttatccagc 360

attcagcatg gacaggttca aaaaataaaa tttcttgata tgggtccact tcaaacatgc 420

gcctacctgt aggaaaaaaa aagagaacat aaatatgccg cgaacagaaa acgtaatgta 480

ctgttctata tataaactgt tcagatcaat cataaattct cagtttcaaa ctttccgctc 540

agccagattg tcgaccatca tcatcatcat cattgagttt gtagccttag acatgactgt 600

tcctcagttc aagttgggca cttacgagaa gaccggtctt gctagattct aatcaagagg 660

atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt ttttatttgt 720

aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga gcttgctcct 780

gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa aatcattcga 840

gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat taagtgagac 900

cttcgtttgt gcggatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac 960

tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag 1020

catactaaat tttccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt 1080

tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat 1140

ttttatcacg tttctttttc ttgaaatttt tttttttagt ttttttctct ttcagtgacc 1200

tccattgata tttaagttaa taaacggtct tcaatttctc aagtttcagt ttcatttttc 1260

ttgttctatt acaacttttt ttacttcttg ttcattagaa agaaagcata gcaatctaat 1320

ctaaggggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac 1380

aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc 1440

gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg 1500

gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag 1560

gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg 1620

tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg 1680

accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac 1740

tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacggcggcc cacgggtccc 1800

aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag ttatgtcacg 1860

cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc 1920

tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat 1980

atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa 2040

aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcaagc tggagaccaa 2100

catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 2160

tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 2220

gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 2280

ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 2340

cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 2400

caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 2460

ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 2520

taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 2580

taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 2640

cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 2700

tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 2760

gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 2820

catgagatc 2829

<210> 13

<211> 1878

<212> DNA

< 213>artificial sequence

<220>

<221> CDS

<222> (1)~(1878)

< 223>gene order and the protein sequence of HSA/PTH (1-34) fusion rotein

<400> 13

gat gca cac aag agt gag gtt gct cat cgg ttt aaa gat ttg gga gaa 48

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu

1 5 10 15

gaa aat ttc aaa gcc ttg gtg ttg att gcc ttt gct cag tat ctt cag 96

Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln

20 25 30

cag tgt cca ttt gaa gat cat gta aaa tta gtg aat gaa gta act gaa 144

Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu

35 40 45

ttt gca aaa aca tgt gtt gct gat gag tca gct gaa aat tgt gac aaa 192

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys

50 55 60

tca ctt cat acc ctt ttt gga gac aaa tta tgc aca gtt gca act ctt 240

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

65 70 75 80

cgt gaa acc tat ggt gaa atg gct gac tgc tgt gca aaa caa gaa cct 288

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro

85 90 95

gag aga aat gaa tgc ttc ttg caa cac aaa gat gac aac cca aac ctc 336

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu

100 105 110

ccc cga ttg gtg aga cca gag gtt gat gtg atg tgc act gct ttt cat 384

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

115 120 125

gac aat gaa gag aca ttt ttg aaa aaa tac tta tat gaa att gcc aga 432

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg

130 135 140

aga cat cct tac ttt tat gcc ccg gaa ctc ctt ttc ttt gct aaa agg 480

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

145 150 155 160

tat aaa gct gct ttt aca gaa tgt tgc caa gct gct gat aaa gct gcc 528

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala

165 170 175

tgc ctg ttg cca aag ctc gat gaa ctt cgg gat gaa ggg aag gct tcg 576

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

180 185 190

tct gcc aaa cag aga ctc aag tgt gcc agt ctc caa aaa ttt gga gaa 624

Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu

195 200 205

aga gct ttc aaa gca tgg gca gta gct cgc ctg agc cag aga ttt ccc 672

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro

210 215 220

aaa gct gag ttt gca gaa gtt tcc aag tta gtg aca gat ctt acc aaa 720

Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

225 230 235 240

gtc cac acg gaa tgc tgc cat gga gat ctg ctt gaa tgt gct gat gac 768

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

245 250 255

agg gcg gac ctt gcc aag tat atc tgt gaa aat caa gat tcg atc tcc 816

Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser

260 265 270

agt aaa ctg aag gaa tgc tgt gaa aaa cct ctg ttg gaa aaa tcc cac 864

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His

275 280 285

tgc att gcc gaa gtg gaa aat gat gag atg cct gct gac ttg cct tca 912

Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser

290 295 300

tta gct gct gat ttt gtt gaa agt aag gat gtt tgc aaa aac tat gct 960

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

305 310 315 320

gag gca aag gat gtc ttc ctg ggc atg ttt ttg tat gaa tat gca aga 1008

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg

325 330 335

agg cat cct gat tac tct gtc gtg ctg ctg ctg aga ctt gcc aag aca 1056

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

340 345 350

tat gaa acc act cta gag aag tgc tgt gcc gct gca gat cct cat gaa 1104

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu

355 360 365

tgc tat gcc aaa gtg ttc gat gaa ttt aaa cct ctt gtg gaa gag cct 1152

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro

370 375 380

cag aat tta atc aaa caa aat tgt gag ctt ttt gag cag ctt gga gag 1200

Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

385 390 395 400

tac aaa ttc cag aat gcg cta tta gtt cgt tac acc aag aaa gta ccc 1248

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

405 410 415

caa gtg tca act cca act ctt gta gag gtc tca aga aac cta gga aaa 1296

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

420 425 430

gtg ggc agc aaa tgt tgt aaa cat cct gaa gca aaa aga atg ccc tgt 1344

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys

435 440 445

gca gaa gac tat cta tcc gtg gtc ctg aac cag tta tgt gtg ttg cat 1392

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His

450 455 460

gag aaa acg cca gta agt gac aga gtc acc aaa tgc tgc aca gaa tcc 1440

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

465 470 475 480

ttg gtg aac agg cga cca tgc ttt tca gct ctg gaa gtc gat gaa aca 1488

Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys

485 490 495

tac gtt ccc aaa gag ttt aat gct gaa aca ttc acc ttc cat gca gat 1536

Leu Val Asn Arg Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp

500 505 510

ata tgc aca ctt tct gag aag gag aga caa atc aag aaa caa act gca 1584

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala

515 520 525

ctt gtt gag ctc gtg aaa cac aag ccc aag gca aca aaa gag caa ctg 1632

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu

530 535 540

aaa gct gtt atg gat gat ttc gca gct ttt gta gag aag tgc tgc aag 1680

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

545 550 555 560

gct gac gat aag gag acc tgc ttt gcc gag gag ggt aaa aaa ctt gtt 1728

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val

565 570 575

gct gca agt caa gct gcc tta ggc tta gga tcc ggt ggt ggt ggt agt 1776

Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Ser Gly Gly Gly Gly Ser

580 585 590

tct gtt tct gaa atc cag ctg atg cac aac ctt ggt aaa cac ctg aac 1824

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Glu Lys His Leu Asn

595 600 605

tcc atg gaa cgt gtt gaa tgg ctg cgt aag aaa ctg cag gat gtc cat 1872

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

610 615 620

aac ttc

Asn Phe

625

Claims (4)

1. method for preparing human serum albumin-human parathyroid hormone, realize through following steps:

(a) design YPS15 ' end homology arm and 3 ' end homology arm amplimer, and from the pichia spp genome, effectively amplify this two homology arm sequences; Wherein be used for amplification YPS1Upstream primer sequence SEQ ID the NO.1:5 '-aagc of 5 ' end homology arm sequence CtgcagAgctccattg cgccaacccc-3 ', the line part does PstThe I restriction enzyme site, downstream primer sequence SEQ ID NO.2:5 '-cggc GtcgacAatctggctg agcggaaagt ttga-3 ', the line part does SalThe I restriction enzyme site,

Be used for amplification YPS1Upstream primer sequence SEQ ID the NO.3:5 '-ccc of 3 ' end homology arm sequence AgatctC acatttcgcg cctgccttcc t-3 ', the line part does BglThe II restriction enzyme site, downstream primer sequence SEQ ID NO.4:5 '-aaa CtgcagG ggtgagcctg tctggccct-3 ', the line part does PstThe I restriction enzyme site;

(b) these two homology arm sequences that obtain in the step (a) are inserted among the carrier pPICZ α B according to sequencing, obtain to be used for the recombinant vectors pPICZ α B-of subsequent homo reorganization YPS1△, sequence is SEQ ID NO.12;

(c) with the recombinant vectors that makes up in the step (b) with single digestion with restriction enzyme after transfection SMD1168; Cultivation screening recon with the selective pressure that contains Zeocin; With the exactness of corresponding positive primer and negative primer checking homologous recombination, correct defective type recon is designated as SMD1168 Yps1△; Wherein negative primer sequence is: 5 '-ctc ctt cca ccg cca cgg c-3 '; 5 '-acc act tga ggc gtc ctt gga-3 '; Positive primer sequence is SEQ ID NO.7:5 '-atg gag gag ttg agg acc-3 ', 5 '-gca aat ggc att ctg aca tcc-3 ';

(d) utilize the protease-deficient host SMD1168 that obtains in the step (c) Yps1△ expresses corresponding proteins lytic enzyme responsive type recombinant protein-human serum albumin-human parathyroid hormone 1-34 fusion rotein, and SEQ ID NO:13 is the gene and the protein sequence of this fusion rotein.

2. a kind of method for preparing human serum albumin-human parathyroid hormone according to claim 1 is characterized in that, in the step (a), provides YPS1The gene order SEQ ID NO.9 of 5 ' end homology arm, YPS1The gene order of the gene order SEQ ID NO.10 of 3 ' end homology arm and coding yapsin 1 YPS1SEQ ID NO.11.

3. a kind of method for preparing human serum albumin-human parathyroid hormone according to claim 1; It is characterized in that; In the step (b), yeast host GS115, SMD1168 and expression vector pPICZ α B buy from Invitrogen Corp.San Diego.California. USA company.

4. the application of human serum albumin-human parathyroid hormone that method according to claim 1 prepares in preparation treatment medicine for treating osteoporosis.

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