Expression vector and strain simultaneously used for in-vivo and in-vitro multiple copies as well as construction method and application of expression vector and strain

Abstract

The invention relates to an in-vivo and in-vitro multi-copy expression vector, a strain and a construction method and application thereof, and belongs to the technical field of gene engineering. The method comprises the following steps: firstly, constructing expression vectors pMChZ-AOX alpha and pMCrZ-AOX alpha by taking a pPIC9K vector as a skeleton, then cloning a gene of a to-be-expressed target protein onto the vector, transforming host bacteria, constructing a recombinant strain for in-vivo and in-vitro multi-copy or high-expression, and carrying out high-expression screening and expression stability verification on the obtained strain. The vector constructed by the invention can give consideration to both in-vitro and in-vivo high-copy screening, and in-vivo high-copy strain screening is carried out by utilizing antibiotics after yeast is transferred, so that the screening time can be shortened; according to the plasmid provided by the invention, the resistance gene can be recycled, and finally an expression strain without the resistance gene is obtained. The invention provides plasmids containing different recombination sites, and the situation that when multiple copies exist on the same site, recombination is carried out, the number of copies is reduced, and then the expression level is reduced can be avoided.

Description

Technical Field

[0001] The present invention relates to a multi-copy expression vector, a strain, and a construction method and application thereof for both in vivo and in vitro use, belonging to the technical field of genetic engineering. Background Art

[0002] Pichia pastoris (K. phaffii) is one of the most widely used yeast species in the pharmaceutical and biotechnology industries. Since the 1990s, Pichia pastoris has become one of the preferred hosts for recombinant protein production. Many research laboratories and companies (e.g., Bisy GmbH, Biogrammatics Inc., Bioingenium, Research Corporation Technologies (RCT), and Validogen GmbH) offer a variety of engineered K. phaffii strains for improving protein expression, secretion, and glycosylation. Invitrogen's well-known "Pichia Expression Kit", which contains two expression strains (GS115 and KM71), two different expression vectors, with (pPIC9) or without (pHIL-S1) a secretion signal, for stably inserting a gene of interest into the AOX1 gene locus, and its comprehensive user guide makes it the first choice for global laboratories when attempting to use Pichia pastoris as an expression host.

[0003] There are many factors affecting the protein expression level in Pichia pastoris, including the codon usage of the target gene, gene copy number, vector integration site, secretion signal, and culture conditions, etc. To improve the expression level, a common strategy is to screen strains containing a multi-copy target gene expression cassette, and currently, several researchers have developed methods to increase the copy number, including multiple sequential transformations using different selection markers, constructing multi-copy gene cassette vectors, and directly screening for high-resistant recombinant transformants or multi-copy clones after single transformation, etc.

[0004] When expressing recombinant proteins in Pichia pastoris, increasing the copy number of the target gene is the first approach considered to enhance the expression level of recombinant proteins. The PpHIS4 on Invitrogen's pPIC9 and pPIC9K (containing a secretion signal) can be used to complement the growth defect of His4-deficient yeast. The Kan resistance gene can be used to screen for expression strains with multiple copies of the integrated target gene. However, this resistance gene cannot be directly used for screening on resistance plates. Instead, screening needs to be carried out first on auxotrophic plates, and high-copy strains only account for 1-10% of His+ transformants. High-throughput screening is required to obtain a substantial number of target strains. The pPICZ series of plasmids from Invitrogen improve the screening efficiency. After electroporation, high-concentration resistance plates can be directly spread, making it easy to screen for high-copy strains. It is suitable for constructing high-copy strains in vivo, but spreading on antibiotic plates with too high a concentration during screening can easily lead to too few transformants.

[0005] In addition to high-copy in vivo, constructing expression plasmids with multiple copies of the target gene in vitro is also a commonly used method. Invitrogen's pAO815 can be used to construct multi-copy plasmids in vitro using isocaudamers. The expression strains constructed by this method can better correspond to the relationship between the expression level and the copy number of the target gene. However, the operation of constructing multi-copy plasmids in vitro is relatively cumbersome, and large plasmids may be difficult to transform. Additionally, the recombination sites of existing commercial vectors are repeated. If different resistance genes are used for screening, homologous recombination may need to be carried out at the same site, which can lead to a decrease in copy number due to the "looping out" of the target gene. Moreover, the resistance genes used in expression plasmids will remain in the engineered strains. In particular, using resistance genes for screening increases the copy number of the resistance genes while increasing the copy number of the target gene, which may be questioned in some application fields and may be harmful to the environment.

[0006] Although several commonly used vectors have their respective advantages, they also have defects in use. For example, the self-promoter before the KanR gene on pPIC9K is too weak, and after electroporation, it cannot be directly spread on resistance plates for screening. When used for PTVA (posttransformational vector amplification), the copy number of the target gene in the screened strains is also lower than that when Zeocin is used as a screening marker. The BleoR on pPICZαB can be directly used for screening on resistance plates after electroporation and is used for constructing high-copy strains in vivo. Moreover, this plasmid is relatively small and can be used to construct high-copy plasmids in vitro. However, this plasmid has fewer recombination sites and can only be recombined at the AOX1 promoter. pAOX815 can be used to construct high-copy plasmids in vitro, facilitating the correspondence between the copy number and the expression level. However, this plasmid is already 7.7 kb, and the constructed copy number is limited.

[0007] In summary, the existing commonly used expression vectors cannot balance high-copy construction in vivo and in vitro, and the recombination sites are single. The plasmids screened using antibiotics cannot recycle the selection markers. Therefore, an expression vector that can balance high-copy construction in vivo and in vitro and overcome the above defects is an actual requirement for research and production in the current technical field. Summary of the Invention

[0008] The purpose of the present invention is to overcome some technical problems existing in the prior art. The present invention provides a multi-copy expression vector, strain, and their construction methods and applications for both in vivo and in vitro use. The present invention aims to construct a plasmid that can balance high-copy construction in vivo and in vitro and can recycle the resistance gene, for quickly constructing high-copy expression strains and removing the resistance gene.

[0009] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0010] The present invention provides a multi-copy expression vector for both in vivo and in vitro use. The expression vector uses the pPIC9K vector as the backbone, and replaces the KanR resistance gene, Bom, and AOX1 3’fragment element region in pPIC9K with a replacement fragment A; deletes BamHⅠ located upstream of αMF in pPIC9K by site-directed mutagenesis, and introduces a BamHⅠ recognition site downstream of AOX1tt. The sequences of other vector elements remain unchanged.

[0011] The multi-copy expression vector for both in vivo and in vitro use is named pMChZ-AOXα, where h represents that the recombination site is PpHIS4, and Z represents that the selection marker is BleoR.

[0012] Among them, both ends of the sequence of the replacement fragment A contain lox71 and lox66 sequences. There is an ori upstream of lox71, and a Cre transcription unit inside. The Cre gene is driven by a pAOX1 promoter mutated at the SacⅠ restriction site. There are prokaryotic and eukaryotic promoters upstream of BleoR, and a prokaryotic promoter upstream of AmpR.

[0013] Furthermore, the nucleotide sequence of the replacement fragment A in the expression vector is as shown in SEQ ID No:1, replacing 4648 - 9221bp of the pPIC9K vector.

[0014] Furthermore, the BamHⅠ located upstream of αMF in pPIC9K deleted by site-directed mutagenesis is in the 938 - 943bp region, and the BamHⅠ recognition site introduced downstream of AOX1tt is in the 1583 - 1584bp region.

[0015] According to an embodiment of the present invention, the PpHIS4 (carried in pPIC9K) of the expression vector pMChZ-AOXα is replaced by substitution fragment B to construct a new expression vector named pMCrZ-AOXα, where r represents that the recombination site is RGI2; further, the sequence of the substitution fragment B is as shown in SEQ ID No:2.

[0016] The present invention also provides the application of the expression vector in constructing multi-copy or high-expression strains in vivo and in vitro.

[0017] The present invention also provides a recombinant expression strain, which comprises the expression vector described above.

[0018] Further, the host bacteria of the recombinant expression strain include yeast, preferably Pichia pastoris, more preferably X33 or GS115.

[0019] The present invention also provides a construction method for simultaneously constructing multi-copy or high-expression strains in vivo and in vitro, the method comprising:

[0020] S1. Cloning the coding gene of the target protein to be expressed into the EcoRⅠ or NotⅠ site of the expression vector according to any one of claims 1-6 to obtain a recombinant expression vector;

[0021] S2. Linearizing the obtained recombinant expression vector and then transforming the host bacteria to obtain a recombinant expression strain;

[0022] S3. Verifying the expression stability by inducing and screening the strains with high expression levels in deep-well plates from the obtained recombinant expression strains;

[0023] S4. Using the strains screened in S3 as the starting strains, and repeating steps S1, S2 and S3;

[0024] Among them, the expression vectors used in steps S1 and S4 are different.

[0025] Among them, the coding gene of the target protein can also be digested with enzymes and then cloned into the BamHⅠ site of pMChZ-POI and / or pMCrZ-POI multiple times to obtain an expression vector containing multiple copies of the coding gene of the target protein.

[0026] At least two transformations with different expression vectors are required when the recombinant expression vector is linearized and then transformed into the host bacteria.

[0027] The method for screening by deep-well plates includes:

[0028] On the first day, the transformants were inoculated into deep-well plates containing 250 μL / well of BMD1 (0.3% K2HPO4, 1.18% KH2PO4, 1.34% YNB, 1% glucose, 4E-05% biotin), and cultured at 800 rpm and 30 °C until the morning of the fourth day. Then, 250 μL / well of BMM2 (0.3% K2HPO4, 1.18% KH2PO4, 1.34% YNB, 1% methanol, 4E-05% biotin) was added. At noon and in the afternoon, 25 μL / well of BMM20 (0.3% K2HPO4, 1.18% KH2PO4, 1.34% YNB, 10% methanol, 4E-05% biotin) was added, and the interval between two additions of the induction medium was 4 h. On the fifth day, 25 μL / well of BMM20 was added twice, with an interval of 8 h. Samples were collected in the morning of the sixth day, and the induction time for the strains was 48 h.

[0029] The method for verifying the expression stability includes:

[0030] The overnight culture broth was transferred to YPD medium at a volume ratio of 1 / 50, and transferred every 24 h. The broth transferred for the third time was induced according to the Pichia pastoris expression operation manual of invitrogen. Subsequent continuous transfers were carried out, and after induction of the broth transferred for the sixth and ninth times, the expression level was verified by SDS-PAGE.

[0031] The present invention also provides the application of the recombinant expression strain in the preparation of a target gene or protein with multiple copies or high expression levels.

[0032] In the present invention, the Cre expression gene may not be located in the expression plasmid, but expressed with a free plasmid. After constructing the expression strain, the Cre recombinase was expressed with a free plasmid to recover the resistance gene.

[0033] The recombination site of the present invention can be replaced with other genes in Pichia pastoris, such as ARG, ENO, TRP2, etc., and the selection marker can be replaced with KanR, HygR, NrsR, etc., and all are applicable to this method.

[0034] The beneficial effects of the present invention:

[0035] In the present invention, two multi-copy expression vectors for in vivo and in vitro use were constructed with pPIC9K as the backbone. The constructed expression vectors can quickly construct high-expression strains and are multi-copy expression vectors suitable for industrial production.

[0036] The present invention uses Pichia pastoris as the host bacterium and successfully and rapidly constructs a recombinant bacterium with high expression of the target gene. The plasmid constructed in the present invention can take into account the advantages of high-copy screening both in vitro and in vivo. It is possible to first construct a multi-copy expression plasmid in vitro and then use antibiotics to screen for high-copy strains in vivo after transferring it into yeast, which can shorten the screening time. The expression vector provided by the present invention can recycle the resistance gene, and finally obtain an expression strain without the resistance gene.

[0037] The expression vector provided by the present invention contains different recombination sites, which can avoid the decrease in copy number and thus the decrease in expression level caused by "looping out" during recombination when there are multiple copies at the same site, and can significantly improve the expression level of the target gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the map of the starting vector pPIC9K.

[0039] Figure 2 It is the map of the modified pMChZ-AOXα.

[0040] Figure 3 It is the map of the modified pMCrZ-AOXα.

[0041] Figure 4 It is the screening process of the high-expression strain in the present invention.

[0042] Figure 5 It is the screening process of the deep-well plate in the present invention.

[0043] Figure 6 It is the schematic diagram of the evaluation process of the expression stability of the strain.

[0044] Figure 7 It is the verification result of the constructed pMChZ-(M6)2 after digestion with NcoI by gel electrophoresis. In the figure, the lanes from left to right are the undigested plasmid, the digested plasmid, and the Marker. The sizes of the Marker from top to bottom are 10000, 8000, 6000, 5000, 4000, 3000, 2000, 1000, and 500 bp respectively.

[0045] Figure 8 It is the verification result of pMChZ-M6 after digestion with EcoRI and NcoI by gel electrophoresis. In the figure, the lanes from left to right are the undigested plasmid, the digested plasmid, and the Marker. The sizes of the Marker from top to bottom are 10000, 8000, 6000, 5000, 4000, 3000, 2000, 1000, and 500 bp respectively.

[0046] Figure 9The verification results of pMCrZ-M6 digested with EcoRI and NcoI were verified by gel electrophoresis. In the figure, the lanes from left to right are the undigested plasmid, the digested plasmid, and the Marker. The sizes of the Marker from top to bottom are 10000, 8000, 6000, 5000, 4000, 3000, 2000, 1000, and 500 bp respectively.

[0047] Figure 10 The SDS-PAGE results of M6 after multiple rounds of electrotransformation. Among them, Figure a shows the screening results of the transformant X33 / M6-1# in the deep well plate obtained after electrotransforming pMChZ-(M6)2 into X33. The control is the strain obtained by high-expression screening of the pPIC9K expression plasmid (denoted as the production strain in the figure). The numbers and letters in the figure represent the corresponding strains in the deep well plate during screening. The circled numbers in front represent the numbers of the deep well plates. The circled number label is omitted for the 1st plate; Figure b shows the rescreening results of the deep well plate after streaking the strains with high expression in a; Figure c shows the flask induction results of the strains with high expression and good consistency in b; Figure d shows the screening results of the transformant X33 / M6-2# in the deep well plate obtained after electrotransforming pMChZ-M6 linearized with SacI into X33 / M6-1#; Figure e shows the screening results of the transformant X33 / M6-3# in the deep well plate obtained from the high-expression strain screened from the electrotransformation of pMCrZ-M6 into X33 / M6-1#; Figure f shows the flask induction results of the strains with the highest expression in different batches.

[0048] Figure 11 The verification results of the expression stability of M6-related strains. Among them, Figure a shows the SDS-PAGE results of the strain supernatant, and Figure b is the result of plotting after measuring the UV of the corresponding strain supernatant and quantifying the protein; the 1 st 、3 rd 、6 th 、9 th represent the transfer times, as Figure 6 shown; 4D, 10D, 3D, and 10F in Figure a correspond to X33 / M6-1#-ZEO S 4D, X33 / M6-1#-ZEO S 10D, X33 / M6-1#-ZEO S 3D, X33 / M6-2#-ZEO S 10F in Figure b.

[0049] Figure 12 The copy number of M6-related strains.

[0050] Figure 13 The results of colony PCR before induction (a) and colony PCR after recovery of the screening marker (b).

[0051] Figure 14The SDS-PAGE results after multiple rounds of electroporation of M4; Figure a shows the screening results of the deep well plate of the transformant X33 / M4-1# obtained after electroporating pMChZ-(M4)2 into X33, and the control is the strain obtained by high-expression screening with the pPIC9K expression plasmid (the production strain in the figure); Figure c shows the screening results of the deep well plate of the transformant X33 / M4-2# obtained after linearizing pMChZ-M4 with SacI and then electroporating it into X33 / M4-1#; Figure b shows the screening results of the deep well plate of the transformant X33 / M4-3# obtained after electroporating pMCrZ-M4 into X33 / M4-1#; Figure d shows the shake flask induction results of the strains with the highest expression levels in different batches.

[0052] Figure 15 It is a report on the copy number of the target gene M6 detected by digital PCR for the strains screened in Example 2 and the control strain. Detailed implementation mode

[0053] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the preferred embodiments of the present invention are described in detail below. However, the following embodiments do not limit the protection scope of the present invention.

[0054] In the embodiments of the present invention, those not described in detail are all completed by conventional experimental methods. For the processes involved in the embodiments that are not described in detail, those skilled in the art can understand and easily implement them according to the product instructions or the basic knowledge in the art, so they will not be described in detail here.

[0055] For each culture medium or other reagent materials involved in the present invention, if not otherwise specified, they are all prepared conventionally or purchased.

[0056] The pPIC9K vector involved in the embodiment was purchased from Invitrogen, product number: V17520, and its sequence information is shown in SEQ.ID.No: 7. It can also be downloaded from the website www.invitrogen.com. The vector map in the vector product instruction is as Figure 1 shown. The size of this vector is 9276bp, and the screening marker is HIS4. The main characteristic elements and their positions of the vector include:

[0057] 5’AOX1 promoter fragment (5’-end AOX1 promoter fragment): bases 1-948;

[0058] α-Factor secretion signal(s) (α-factor secretion signal): bases 949-1218;

[0059] Multiple cloning site (multiple cloning site): bases1192-1241;

[0060] 3’AOX1 transcription termination (TT) (3’-end AOX1 terminator): bases 1253 - 1586; HIS4 ORF

[0061] (corresponding to PpHIS4 in the figure): bases 4514 - 1980;

[0062] Kanamycin resistance gene (KanR, kanamycin resistance gene): bases 5743 - 4928;

[0063] 3’AOX1 fragment (3’-end AOX1 fragment): bases 6122 - 6879;

[0064] pBR322 origin (pBR322 replication origin, represented as ori in the figure): bases 7961 - 7288,;

[0065] Ampicillin resistance gene (AmpR, ampicillin resistance gene): bases 8966 - 8106.

[0066] Example 1:

[0067] (1) Construct an expression plasmid that can be used for both in vitro and in vivo multi-copy

[0068] Synthesize the SEQ ID No:1 sequence and replace the 4648 - 9221bp of the pPIC9K vector sequence, that is, replace the KanR resistance gene, Bom and AOX1 3’fragment elements in pPIC9K with the replacement fragment SEQID No:1; delete BamHⅠ(938 - 943bp) upstream of αMF (i.e., α-factor secretion signal) in pPIC9K by site-directed mutagenesis, and introduce a BamHⅠ recognition site by site-directed mutagenesis between 1583 - 1584bp downstream of AOX1tt (also known as AOX1 transcription termination or AOX1 termination), and the other vector element sequences remain unchanged.

[0069] Among them, the synthesized SEQ ID No:1 sequence contains lox71 and lox66 sequences at both ends. There is an ori upstream of lox71 and a Cre transcription unit inside. The Cre gene is driven by the AOX1 promoter with a SacⅠ restriction site mutation; there are prokaryotic and eukaryotic promoters upstream of BleoR, which can be used for screening in prokaryotes and eukaryotes, and there is a prokaryotic promoter upstream of AmpR, which can be used for prokaryotic screening.

[0070] The plasmid pMChZ-AOXα was constructed, where h represents that the recombination site is PpHIS4 and Z represents that the selection marker is BleoR.

[0071] This plasmid can express Cre recombinase upon methanol induction, catalyze the recombination of lox71 and lox66, and realize the recovery of the resistance gene. BamHⅠ located after AOX1tt can be used for constructing in vitro multi-copy plasmids with isocaudamers. The original plasmid pPIC9K and the plasmid map constructed in the present invention are as Figure 1 and Figure 2 shown.

[0072] The meanings of each part in SEQ ID No:1 are explained as follows:

[0073] The lowercase letter part is ori, the 5'-end with a gray background is lox71, and the 3'-end with a gray background is lox66; the single underline _ is AmpR, the double underline is pAmpR (AmpR promoter), the wavy line is the pAOX1 promoter with a mutated SacⅠ site, the dotted line is the Cre recombinase gene, the heavy dot is AOX1tt, the _ thick underline is pTEF1 (corresponding to TEF1 promoter in the figure), the lowercase letters with a wavy line are pEM7 (corresponding to EM7 promoter in the figure), the italic lowercase letters are BleoR, and the lowercase letters with an underline _ are CYC1tt (corresponding to CYC1 terminator in the figure). The sequence information of SEQ ID No:1 is as follows:

[0074]

[0075]

[0076]

[0077] The SEQ ID No:2 sequence was synthesized to replace PpHIS4 on pMChZ-AOXα, and the plasmid pMCrZ-AOXα was obtained, where r represents that the recombination site is RGI2, and the map is as Figure 3 shown.

[0078] The sequence information of SEQ ID No:2 is as follows:

[0079]

[0080] Verify the high expression of the constructed high-expression vector.

[0081] (2) Clone the target gene into the EcoRⅠ and NotⅠ sites of pMChZ-AOXα and pMCrZ-AOXα respectively to obtain pMChZ-POI (Protein of Interest) and pMCrZ-POI; the POI transcription unit is digested with BglⅡ and BamHⅠ and then cloned into the BamHⅠ site of pMChZ-POI and pMCrZ-POI to obtain two copies of the expression vector. According to Figure 4 the shown process, construct and screen the expression strain. At least perform electroporation twice. Expression plasmids with different recombination sites need to be used. The actual operation can be adjusted. The copy number of POI on different plasmids can be determined by yourself, and the electroporation order can also be changed.

[0082] It should be noted here that: since the recombination site is a sequence homologous to the Pichia pastoris genome on the plasmid, after linearization with restriction endonuclease, it is electroporated into yeast and homologous recombination occurs at this site. In the examples of the present invention: the recombination site in the first round of electroporation is PpHIS4, and the recombination site in the second round of electroporation is RGI2. The same recombination site as the previous time cannot be used, nor can pAOX1 on the original plasmid be used, that is, different plasmids are required. In the examples of the present invention, an additional experiment was conducted to use plasmids with the same plasmid backbone for two rounds of electroporation as a comparison. The results showed that the expression level decreased.

[0083] (3) According to Figure 5 the shown process, perform deep-well plate screening. Inoculate the transformants into a deep-well plate containing 250 μL / well of BMD1 (K2HPO4 0.3%, KH2PO4 1.18%, YNB 1.34%, glucose 1%, biotin 4E-05%) on Friday of the previous week (the first day), and culture at 800 rpm and 30 °C until Monday morning (the fourth day). Then add 250 μL / well of BMM2 (K2HPO4 0.3%, KH2PO4 1.18%, YNB 1.34%, methanol 1%, biotin 4E-05%). Subsequently, add 25 μL / well of BMM20 (K2HPO4 0.3%, KH2PO4 1.18%, YNB 1.34%, methanol 10%, biotin 4E-05%) at the time shown in Figure 5 .

[0084] As an alternative, if the transformants are obtained on Monday (the first day), shorten the BMD1 culture time. Inoculate the transformants into the deep-well plate on Monday (the first day) and start induction on Wednesday (the third day). The supplementation frequency and volume remain unchanged. The induction time for methanol-utilizing strains is 48 h.

[0085] (4) The strains with ideal expression levels screened out are subjected to expression stability verification, such as Figure 6 shown in: The overnight culture broth was transferred to a 100 mL shake flask containing 10 mL of fresh YPD medium at a volume ratio of 1 / 50, and transferred every 24 h. The broth of the third transfer was induced according to the Pichia pastoris expression operation manual of Invitrogen (Invitrogen: Pichia Expression Kit USERGUIDE For expression of recombinant proteins in Pichia pastoris, Usermanual. 2020). Subsequent continuous transfers were carried out, and the broth of the sixth and ninth transfers was induced, and the expression level was verified by SDS-PAGE.

[0086] The specific induction method is as follows: The broth was diluted by an appropriate multiple to measure OD600. An appropriate amount of broth was taken according to the initial induction broth OD600 = 10, centrifuged at 3000 rpm for 3 min, and the cell pellet was resuspended in BMM2 medium, cultured at 220 rpm and 30 °C, and methanol was added once in the morning and evening every day until the final concentration was 1%, and the samples were collected after 48 h of induction to detect the expression level.

[0087] All relevant sequences were commissioned to be synthesized by GenScript Biotech Corporation.

[0088] Example 2: Rapid screening of high-expression strains based on M6 using the vector constructed by the present invention

[0089] (1) Construction of relevant expression plasmids

[0090] According to the method in Example 1, the expression plasmids pMChZ-AOXα and pMCrZ-AOXα were constructed.

[0091] M6 (SEQ ID No: 6 in the original patent) in the patent with the publication number CN116082493B was optimized according to the Pichia pastoris preferred codons after adding the LEKR sequence at the N-terminus, and EcoRⅠ and NotⅠ restriction sites were added to the 5' end and 3' end respectively, and then cloned into the EcoRⅠ and NotⅠ restriction sites of pMChZ-AOXα and pMCrZ-AOXα to obtain pMChZ-M6 and pMCrZ-M6.

[0092] The M6 transcription unit was amplified using primers F: 5'-ATAGACGCAGATCGGGAAC-3' and R: 5'-TTTAACTGTGATAAACTACCGC-3', digested with BglⅡ and BamHⅠ and then cloned into the BamHⅠ site of pMChZ-M6 to obtain the expression plasmid pMChZ-(M6)2 containing 2 copies of M6.

[0093] The constructed plasmid was verified by gel electrophoresis after digestion with EcoRI and NcoI. The verification results are as Figures 7 - 9 shown. As can be seen in the figure, pMChZ-(M6)2, pMChZ-M6, and pMCrZ-M6 were verified by NcoI digestion, and their sizes were as expected; Figures 7 - 9 In it, the lanes from left to right are the undigested plasmid, the digested plasmid, and the Marker. The sizes of the Marker from top to bottom are 10000, 8000, 6000, 5000, 4000, 3000, 2000, 1000, and 500 bp respectively.

[0094] The sequence of M6 after adding the LEKR sequence at the N-terminus is shown in SEQ ID No:3:

[0095] LEKRGPPGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGPQGVKGESGKPGANGLSGENGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGPQGVKGESGKPGANGLSGENGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGPQGVKGESGKPGANGLSGENGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGPQGVKGESGKPGANGLSGENGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGPQGVKGESGKPGANGLSGENGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGG

[0096] The sequence of M6 after adding the LEKR sequence at the N-terminus and optimized according to the preferred codons of Pichia pastoris, with EcoRⅠ and NotⅠ restriction sites added at the 5' and 3' ends respectively, is shown in SEQ ID No:4: (The 5' end with a gray background is EcoRⅠ, and the 3' end with a gray background is NotⅠ)

[0097]

[0098] (2) Construction and screening of expression strains

[0099] The expression plasmids pMChZ-(M6)2 and pMChZ-M6 were linearized with SalⅠ and SacⅠ respectively, precipitated with ethanol and dissolved in an appropriate amount of water to a concentration of about 300 ng / μL.

[0100] Using X33 (Cat. No.: C18000, Invitrogen TM ) as the starting strain, the competent cells were prepared using the method described by Lin-Cereghino (Lin-Cereghino et a L, 2005). 40 μL of competent cells were mixed with 4 μL (1.2 μg) of the pMChZ-(M6)2 plasmid linearized with SalI. Electroporation conditions: V = 1.5 kV, C = 25 μF, PC = 200 Ω. After electroporation, 1 mL of YPD:1 M sorbitol (the volume ratio of the two is 1:1) was added and incubated at 30 °C for 4 h, and then spread on YPDZ (YPD plus Zeocin antibiotic 300 μg / mL) and YPDZ (YPD plus Zeocin antibiotic 500 μg / mL) plates. The grown transformants were designated as X33 / M6-1#-Zeo R (Zeo R is the default).

[0101] In the examples of the present invention, the naming rule of the obtained transformants is: starting strain name / POI - number of electroporation times# - sensitivity to screening antibiotics. If the screening marker is not recovered, the description of antibiotic sensitivity will be omitted. Here, Zeo is the abbreviation of Zeocin, S (sensitive) represents sensitive, and R (resistant) represents resistant. In addition, in the labeling of strains, labels such as 4D and 3E represent positions in deep well plates. In the strain numbering, the positions are not specifically defined and are only used to mark the positions in the plate. The horizontal direction of the deep well plate is labeled with Arabic numerals 1-12, and the vertical direction is labeled with capital letters A-H. The circled number before the marker (such as 4D, 3E, etc.) indicating the position of the indicator strain in the well plate represents the 96-well plate number, and the circled number after the marker represents different monoclonal colonies of the same strain during shake flask induction; for example: Figure 10 In a, X33 / M6-1#-③4D represents the transformant at the 4D well position of the third 96-well or deep well plate after electroporating X33 with pMChZ-(M6)2, Figure 10 In c, X33 / M6-1#-4D① represents the first monoclonal colony after streaking and subculturing in a shake flask after rescreening in a deep well plate.

[0102] The transformants were inoculated into deep well plates. Meanwhile, X33 transformed with the unmodified vector pPIC9K was used as a control. The deep well plates were induced for 48 h according to the method described in Example 1. The supernatant was collected by centrifugation, and the absorbance at 215 nm (A215) and 225 nm (A225) was measured. The total protein content in the supernatant was calculated according to the empirical formula C (mg / mL) = 0.144 × (A215 - A225). The supernatants of the strains with the top 5% total protein concentrations were taken for SDS-PAGE. The results were as follows Figure 10 a. The strains with high expression levels were streaked, and the expression homogeneity was verified using deep well plates. Six wells in one column of a 96-well plate were inoculated with each strain. The strain with the highest UV quantification value for each type of strain was selected for SDS-PAGE. The results were as follows Figure 10 b. Subsequently, the strain X33 / M6-1#-4D-Zeo with high expression and high homogeneity was selected S (The screening marker was recovered, and it was sensitive to antibiotics, marked as S. This strain was X33 / M6-1#-Zeo R the screened strain with high expression) was verified using shake flasks or used as the starting strain for the next round of electroporation. Figure 10 c is Figure 10 the verification result of the strain with the highest expression level in b after streaking again using shake flasks.

[0103] Using X33 / M6-1#-4D-Zeo S as the starting strain, pMChZ-M6 linearized by SacⅠ was electroporated, that is, linearized at pAOX1 of this plasmid. After the plasmid was electroporated into the strain, recombination occurred at the AOX1 promoter. The constructed strain was named X33 / M6-2#. The deep well plates were induced, and the strains with the top-listed expression levels were selected by UV quantification. The supernatant was collected by centrifugation for SDS-PAGE. The results were as follows Figure 10 d. It was found that when repeated electroporation was performed with the same plasmid backbone, the expression level decreased (the starting strain was X33 / M6-1#-4D-Zeo s , Figure 10 in d, pMChZ-M6 linearized by SacI was electroporated into X33 / M6-1#-4D-Zeo s to obtain X33 / M6-2#, and the expression decreased).

[0104] Using X33 / M6-1#-4D-Zeo S as the starting strain, pMCrZ-M6 linearized by SpeⅠ was electroporated, and the recombination site was changed to RGI2. The constructed strain was denoted as X33 / M6-3#. The deep well plates were induced, and the strains with the top-listed expression levels were selected by UV quantification. The supernatant was collected by centrifugation for SDS-PAGE. The results were as follows Figure 10 e. Some strains with increased expression levels were obtained.

[0105] The strains screened from different batches of deep well plates were verified using shake flasks. The results were as follows Figure 10 f. The results were consistent with those of the deep well plates.Figure 10 a, 10b, 10d, and 10e are the screening results of deep well plates. 10f is the verification of the strain with the highest expression level screened from different batches of deep well plates in shake flasks. In Figure f, the expression level of X33 / M6-1# is about twice that of the control strain (the strain screened by spotting the plasmid constructed with pPIC9K), the expression level of X33 / M6-2# decreases significantly, and the expression level of X33 / M6-3# is higher than that of X33 / M6-1#. Further explanation shows that when using expression plasmids with different recombination sites for transformation, the expression level of the target gene in the obtained strains increases, but when using expression plasmids with the same recombination site for transformation, the expression level of the target gene in the obtained strains will decrease.

[0106] The method for preparing competent cells in this example is as follows:

[0107] Pick a fresh colony (no more than 2 weeks old) grown by streaking on a plate into a centrifuge tube / Erlenmeyer flask containing 5 mL of YPD, and culture overnight at 30 °C and 220 rpm; transfer the overnight culture to 50 mL of YPD to make A600 0.15 - 0.20, and place it in a sufficiently large flask to provide good aeration; culture the yeast at 30 °C and 220 rpm until A600 reaches 0.8 - 1.0; centrifuge at 500×g for 5 minutes at room temperature and discard the supernatant; prepare a cell treatment solution by adding 1 mL of 1.0 M DTT to 9 mL of BEDS solution [10 mM bicine-NaOH, pH 8.3, 3% (v / v) ethylene glycol, 5% (v / v) DMSO, and 1 M sorbitol], and resuspend the cell pellet in 10 mL of ice-precooled cell treatment solution; culture the cell suspension at 30 °C and 100 rpm for 5 minutes; centrifuge again at 500×g for 5 minutes at room temperature, and resuspend the cells in 1 mL (0.02 times the volume of the culture in step 2) of BEDS solution without DTT; the prepared competent cells can be directly used for electroporation, or the cells can be aliquoted and stored frozen at -80 °C. The competent cells can be stored at this temperature for at least 6 months.

[0108] (3) Verification of expression stability

[0109] The expression stability of the expression strains screened from different batches was verified by the method described in step (4) of Example 1, and the results are as Figure 11As shown in the figure, 1st, 3rd, 6th, and 9th in the figure represent the number of transfer times. Different transformants (expression strains) obtained by multiple electroporations with the same plasmid were used to verify the expression stability of each strain. Among them, Figure a shows the SDS-PAGE results of the strain supernatant, and Figure b shows the results of measuring UV of the corresponding strain supernatant and plotting after quantifying the protein; it can be seen from the figure that not all strains are stably expressed. Since there are no free plasmids in Pichia pastoris, the expression vector is usually integrated into the genome by homologous recombination to construct the expression strain, and the obtained strain is generally considered to be genetically stable. In fact, it is necessary to verify the expression stability when evaluating the strains used for production.

[0110] (4) Detection of the copy number of M6-related strains

[0111] Figure 10 The strains screened from different batches in f and the control production strain were entrusted to Sangon Biotech (Shanghai) Co., Ltd. to detect the copy number of the target gene M6 using digital PCR. The PCR instrument was the T100 of BIO-RAD. According to the estimated yeast genome size of 9.4 Mb, the mass of 1 copy of the genome was 10.33E-06 ng, and the copy number of the genome in 1 μL of the template was calculated. In the report ( Figure 15 ) The ratio of the final copy number of 1 μL of DNA to the copy number of the genome in 1 μL of the template is the copy number of the target gene in yeast. The ratio of the copy number of M6 in each strain to the copy number of M6 in the production strain was plotted. As Figure 12 shown, the copy number detection results were consistent with the SDS-PAGE results. Repeated electroporation with the same plasmid backbone led to the loss of the target gene.

[0112] This example shows that repeated electroporation with the same plasmid reduced the copy number of the target gene. The plasmid backbones of pMChZ-(M6)2 and pMChZ-M6 were the same, and the expression levels generally decreased after repeated electroporation.

[0113] Using this method, the 2-copy plasmid pMChZ-(M6)2 was first constructed in vitro. After electroporation, 3 96-well plates were screened, and multiple strains with higher expression levels than the control (the control used the pPIC9K plasmid and was screened by spotting on plates in the traditional method, and the strains screened from 4 96-well plates) could be obtained from each plate. The expression levels of X33 / M6-2# obtained after electroporation of pMChZ-M6 all decreased. In Figure 11 there are partial protein concentrations quantified by UV. When induced for the first time, the control expression level was 1 mg / mL, and the three strains of X33 / M6-1# were all around 1.8 mg / mL. One strain of X33 / M6-2# was selected as a representative, and the expression level was also 1 mg / mL when induced for the first time.

[0114] (5) Verification of resistance gene recovery

[0115] After induction in deep well plates, the strains were streaked on YPD and YPDZ (100 μg / mL) simultaneously. The strains that could not grow on the YPDZ plate but grew normally on the YPD plate were the strains with the recovered selection marker. One 2-mm monoclonal of the recovered marker strain was taken and put into a PCR tube containing 10 μL of 0.02 M NaOH solution. After vortexing to suspend the cells, it was centrifuged instantaneously. The PCR tube was placed on a PCR instrument, taken out after 10 min at 99 °C, allowed to cool to room temperature, and centrifuged instantaneously. The supernatant was the PCR template, and PCR identification was performed using the universal primers 5’AOX and 3’AOX. Premix Taq TM was used for PCR, and the reaction conditions were: 94 °C for 2 min; 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min, for 30 cycles; 72 °C for 2 min. The results were as Figure 13 , and the selection marker was successfully recovered. Figure 13 In a, the transformants were verified using the universal primers 5’AOX, 3’AOX and 5’AOX, M6-1veri R, which proved to contain the target gene M6 and AOX1, Cre. After recovering the selection marker, as Figure 13 shown in b, there was no Cre band, and only AOX1 remained.

[0116] The primer sequences involved are as follows:

[0117] 5'AOX:GACTGGTTCCAATTGACAAGC

[0118] 3'AOX:GGCAAATGGCATTCTGACAT

[0119] M6-1 veri R:TCCCCGGAAAGTCCATTAGC

[0120] Example 2: Based on M4, high-expression strains were rapidly screened using the vector constructed by the present invention

[0121] (1) Construction of related expression plasmids

[0122] M4 (SEQ ID NO:3 in the original patent) in the patent with the publication number CN 116082493 B was used to construct the expression plasmids pMChZ-M4, pMChZ-(M4)2 and pMCrZ-M4 according to the method in Example 2. The cloning sites and primers were the same as those in Example 2.

[0123] The sequence of M4 after adding the LEKR sequence at the N-terminus is shown in SEQ ID No:5:

[0124] LEKRAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPSGPAGITGAKGPAGPPGMPGPKGSPGPQGVKGESGKPGAQGLSGEKGPPGPQGLPGPAGTAGEPGKDGNPGSDGLPGKDGSPGGKGEKGENGSPGAPGAPGHPGPPGPVGPAGKSGEKGESGPAGPAGAPGPAGSKGAPGPQGPRGDKGETGEKGPAGEKGHRGPPGNPGAPGSPGPAGQQGAIGSPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPSGPAGITGAKGPAGPPGMPGPKGSPGPQGVKGESGKPGAQGLSGEKGPPGPQGLPGPAGTAGEPGKDGNPGSDGLPGKDGSPGGKGEKGENGSPGAPGAPGHPGPPGPVGPAGKSGEKGESGPAGPAGAPGPAGSKGAPGPQGPRGDKGETGEKGPAGEKGHRGPPGNPGAPGSPGPAGQQGAIGSPGPA

[0125] After adding the LEKR sequence to the N-terminus of M4 and optimizing according to the preferred codons of Pichia pastoris, the sequence after adding EcoRⅠ and NotⅠ restriction sites to the 5'-end and 3'-end respectively is shown in SEQ ID No:6 (the 5'-end with a gray background is EcoRⅠ, and the 3'-end with a gray background is NotⅠ):

[0126]

[0127] (2) Construction and screening of expression strains

[0128] The expression plasmid pMChZ-(M4)2 was linearized with SalⅠ and then electrotransformed into X33. The transformants grown on YPDZ were named X33 / M4-1#; X33 / M4-1#-Zeo was obtained by screening out the screening marker from X33 / M4-1# S Using it as the starting strain, pMChZ-M4 linearized with SacⅠ was electrotransformed, and the obtained transformants were named X33 / M4-2#; X33 / M4-1#-Zeo S Using it as the starting strain, pMCrZ-M4 linearized with SpeⅠ was electrotransformed, and the obtained transformants were named X33 / M4-3#. The transformants in deep well plates of different batches were induced, centrifuged, and the supernatant was taken to measure UV. Then, the strains with the top expression levels were selected for SDS-PAGE. The results are as follows Figure 14 , which is consistent with the screening results of M6. When using the same plasmid backbone, even if the recombination site changes, it will also lead to a decrease in the expression level. After changing the recombination site to RGI2, repeating the electrotransformation will not cause a decrease in the expression level even if the expression level cannot be increased

[0129] For the same plasmid, there will be more than one sequence available for homologous recombination. If the same plasmid and the same recombination site are used, it cannot be effectively transferred (repeating the electrotransformation of pMChZ-(M6)2 linearized at PpHIS4 cannot obtain enough transformants). When different recombination sites (changing the recombination site to pAOX1) are selected for the two electrotransformations, it will still lead to plasmid "looping out" and a decrease in the expression level. After changing to pMCrZ-AOXα and changing the recombination site to RGI2, and then performing electrotransformation again, some of the obtained transformants have increased expression levels and will not have a consistent decrease in the expression level like X33 / M6-2# obtained by electrotransforming pMChZ-M6

[0130] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention

Claims

1. A multi-copy expression vector for both in vivo and in vitro use, characterized in that, The expression vector uses the pPIC9K vector as a backbone, and replaces the KanR resistance gene, Bom and AOX1 3’fragment element regions in pPIC9K with replacement fragment A; Delete BamHⅠ upstream of αMF in pPIC9K by site-directed mutagenesis, introduce a BamHⅠ recognition site downstream of AOX1tt, and keep the sequences of other vector elements unchanged.

2. The expression vector according to claim 1, wherein Both ends of the sequence of the replacement fragment A contain lox71 and lox66 sequences. There is an ori upstream of lox71, and a Cre transcription unit inside. The Cre gene is driven by the AOX1 promoter mutated at the SacⅠ restriction site. There are prokaryotic and eukaryotic promoters upstream of BleoR, and a prokaryotic promoter upstream of AmpR.

3. The expression vector according to claim 1, characterized in that, The nucleotide sequence of the replacement fragment A is as shown in SEQ ID No:1, replacing 4648-9221bp of the pPIC9K vector.

4. The expression vector according to claim 1, characterized in that, The BamHⅠ upstream of αMF in pPIC9K deleted by site-directed mutagenesis is located in the 938-943bp region, and the introduced BamHⅠ recognition site downstream of AOX1tt is located in the 1583-1584bp region.

5. The expression vector according to any one of claims 1-4, characterized in that, PpHIS4 in the expression vector is replaced by replacement fragment B to construct a new expression vector.

6. The expression vector according to claim 5, characterized in that, The replacement fragment B is as shown in SEQ ID No:

2.

7. Use of the expression vector according to any one of claims 1-6 in constructing multi-copy or high-expression strains in vivo and in vitro.

8. A recombinant expression strain, characterized in that, The strain contains the expression vector according to any one of claims 1-6.

9. The strain according to claim 8, characterized in that, The host bacteria of the recombinant expression strain include yeast, preferably Pichia pastoris, more preferably X33 or GS115.

10. A method for constructing multi-copy or high-expression strains simultaneously in vivo and in vitro, the method comprising: S1. Clone the coding gene of the target protein to be expressed into the EcoRⅠ or NotⅠ site of the expression vector according to any one of claims 1-6 to obtain a recombinant expression vector; S2. Linearize the obtained recombinant expression vector and transform the host bacteria to obtain a recombinant expression strain; S3. Induce and screen the strains with high expression levels in deep well plates of the obtained recombinant expression strains for expression stability verification; S4. Using the strains screened in S3 as the starting strains, repeat steps S1, S2 and S3; Among them, the expression vectors used in steps S1 and S4 are different.

11. The construction method according to claim 10, characterized in that The coding gene of the target protein can also be digested with enzymes and then cloned into the BamHⅠ site of the recombinant expression vector multiple times to obtain an expression vector containing multiple copies of the coding gene of the target protein.

12. The construction method according to claim 10, characterized in that, At least two transformations with different expression vectors are required when the linearized recombinant expression vector is transformed into the host bacteria.

13. Use of the strain according to any one of claims 8-9 or the strain constructed by the method according to any one of claims 10-12 in the preparation of multi-copy or high-expression target genes or proteins.

Images