TWI633186B - Used to enhance the performance of proteins, calories, vectors and viruses - Google Patents

Abstract

The present invention provides a bidirectional promoter performance cassette for enhancing protein expression, the bidirectional promoter performance cassette comprising a first performance cassette and a second performance card operatively coupled in reverse to the first performance cassette cassette. The first performance card includes: a first baculovirus promoter, at least one pulse sequence, a baculovirus GP64 gene sequence, and a terminator; wherein the second performance card sequence comprises: a second rod A viral promoter, a transcription factor, and a terminator. The expression vector comprising the bidirectional promoter can display a large amount of different foreign proteins on the baculovirus envelope for application to different subunit vaccines.

Description

Used to enhance protein performance, calories, vectors and viruses

The present invention relates to a performance cassette, in particular to a performance card which can enhance the performance of a recombinant protein; the invention also relates to a performance vector, in particular to a performance carrier comprising the aforementioned performance cassette and enhancing the performance of the recombinant protein; A recombinant baculovirus, in particular, a recombinant baculovirus comprising the aforementioned expression vector. The invention also relates to a use for enhancing the performance of recombinant proteins, in particular by the use of the aforementioned expression vectors for achieving enhanced expression of recombinant proteins. The invention also relates to a vaccine comprising the recombinant baculovirus as described above.

Insect baculovirus infection of insect cells can produce two types of viruses, one is budded virion (BV), which is extracellular virus (ECV) or nonoccluded virus (NOV). The other is an occluded body-derived virus (OV) or a polyhedra-derived virus (PDVs). In 1983, Smith and Summer found that the deletion of the polyhedrin gene in Autographa californica nucleopolyhedrosis virus (AcMNPV) did not affect the performance of other proteins and viral infection, and the late promoter p10 was used. And polyhedrons (polyhedron) can express a large number of protein properties, using insect cells as bioreactors to produce exogenous proteins. The recombinant baculovirus expression system belongs to the eukaryotic system. In addition to the large-scale production of exogenous proteins, post-translational modification and the biological activity of the produced proteins, the most important thing is that the expressed proteins are A well-folded configuration, highly similar to other eukaryotic proteins, retains the original properties of the protein.

In the recombinant baculovirus expression system, the commonly used cell is the pupal ovarian tissue of Spodoptera frugiperda, which contains two cells, Sf-9 and Sf-21. cell types for higher sensitivity of the AcMNPV; additionally comprising cells BTI-TN-5B1-4 (product of Invitrogen company high Five ^TM), Trichoplusia ni cell sources (Trichoplusia ni). As for the virus used in the baculovirus system, the virus strain which has been developed and used is AcMNPV and Bombyx mori nuclear polyhedrosis virus (BmNPV). There is a difference between the two. AcMNPV has the advantage of using cells to produce exogenous proteins in large quantities; BmNPV is suitable for the mass production of exogenous proteins in larvae. Past studies have demonstrated that performance proteins are well immunogenic and have the potential to prepare subunit vaccines. Such as: Enterovirus 71 (Chung et al. , 2010), avian reovirus subunit vaccine (Lin et al., 2008) and avian influenza vaccine (H5N1) vaccine (Wu et Al., 2011). Although the baculovirus system has many advantages, the medium or additives required for the eukaryotic expression system will increase the unit cost of production, and the protein expression is not as good as E. coli , so prepare a cellular subunit vaccine or other. Immune preparations or factors may not meet the low cost requirements of commercial sales. Therefore, improving the performance of the eukaryotic performance system has become the focus of development.

In view of the shortcomings of commercially available vectors in low yields in eukaryotic systems and their disadvantages for commercial production, it is an object of the present invention to provide a dual expression promoter for increasing the expression of recombinant proteins. The sub-performance card includes a first performance card and a second performance card operatively coupled to the first performance card. The first performance card includes: a first baculovirus promoter, at least one burst sequence (BS), a baculovirus GP64 gene sequence, and a terminator, and the terminator includes, but is not limited to, Simian vacuolating virus 40 poly A (SV40 pA). The second performance cassette comprises: a second baculovirus promoter, a transcription factor and a terminator; the transcription factor is very late factor-1 (VLF-1) The terminator includes, but is not limited to, herpes simplex virus thymidine kinase polyadenylation (HSV tk pA).

Preferably, the first baculovirus promoter comprises a polyhedron promoter (Pph).

Preferably, the baculovirus GP64 gene sequence comprises a signal peptide (SP), a transmembrane domain (TM) and a cytoplasmic domain (CTD).

Preferably, the signal peptide and the transmembrane region of the baculovirus GP64 gene sequence further comprise a plurality of multiple cloning sites (MCS).

Preferably, the baculovirus GP64 gene sequence further comprises a porcine circovirus type II structural coat protein between the signal peptide and the transmembrane region, and the N-terminal 41 amino acid is deleted. [porcine circovirus 2-capsid protein d41 , PCV2-Cap(d41)].

Preferably, the second baculovirus promoter comprises a P10 promoter.

Preferably, the number of the at least one pulse sequence is one, and the pulse sequence and the baculovirus GP64 gene sequence further comprise a first mutation sequence (mutant signal 1, ms1), the pulse sequence and the first The mutated sequence (BSms1) is shown as SEQ ID No: 8.

Preferably, the number of the at least one pulse sequence is one, and the pulse sequence and the baculovirus GP64 gene sequence further comprise a second mutation sequence (mutant signal 2, ms2), the pulse sequence and the second The mutated sequence (BSms2) is shown as SEQ ID No: 11.

According to the present invention, "performance cassette" as used herein refers to a nucleotide sequence produced by recombinant techniques or synthesis, enabling gene expression in a host cell. Performance cards can be embedded in plastids or chromosomes. In general, the expression cassette portion of the expression vector comprises a promoter, a nucleotide sequence to be transcribed, and a polyadenylation. According to the present invention, "bidirectional promoter expression cassette" as used herein refers to a representation cassette containing a nucleotide sequence to be transcribed encoding two or more gene products. Specifically, in the present case, after the first baculovirus promoter of the first performance cassette, the second baculovirus promoter of the second expression cassette is reversely linked.

The present invention further provides a performance vector comprising the above-mentioned bidirectional promoter expression cassette, which facilitates bringing the target antigen fragment to the cell membrane by including the GP64 gene in the expression vector.

In addition, the foregoing expression carrier inserts two pulse sequences (ie, BS2), one pulse sequence and the first mutation sequence BSms1, or one pulse sequence and the second mutation sequence BSms2, etc., downstream of the polyhedrin promoter, and reversely constructs p10 start-up. The sub-expression is infected with the very late transcription factor Vlf-1, and the transcription factor positively regulates the promoters PphBS2, PphBSms1, PphBSms2, and enhances protein translation efficiency.

The present invention further provides a genetically recombinant baculovirus comprising the above-described expression vector, which is formed by transfection into an insect cell as described above.

Preferably, the species of the insect cell comprises, but is not limited to, Sf-9 ( Spoodoptera fugiperda ) cells or Hi-5 (BTI-TN-5B1-4) cells.

The invention further provides the use of a performance vector as described above for enhancing protein expression by transfecting an expression vector into an insect cell.

The invention further provides a vaccine comprising the recombinant baculovirus of the invention as described above for use in generating an immune response in a recipient. Preferably, the recipient is a pig.

The present invention employs ^{Bac-To-Bac ® Baculovirus Expression} System (Invitrogen, Carlsbad, CA, USA) to prepare recombinant baculovirus, while cloning GP64 (containing SP, TM, CTD domain) protein gene such that the carrier includes a surface rendering properties. The exogenous gene is then inserted into the SP and TM of GP64, and the downstream gene expression is initiated by the baculovirus late promoter polyhedron or p10. The expressed fusion protein is displayed on the cell membrane as a trimer and the baculovirus envelope. on. Next, the present invention uses the Sf-9 cell strain as a host, and transfects the constructed Bacmid DNA into the cells to prepare a recombinant baculovirus. Finally, the present invention selects the Hi-5 cell line as a host, and the recombinant baculovirus infects the host into the cell to express a large amount of the target protein PCV2-Cap (d41), and the surface of the infected cell presents GP64PCV2Cap (d41) protein, and then rapidly The cells are lysed by freeze-thaw method to produce a large number of cell debris, and the fragments contain a recombinant protein which can effectively induce an immune reaction, and the cell lysate is a PCV secondary unit vaccine.

The invention is exemplified by the following examples, which are not to be construed as limiting the scope of the invention.

Preparation Example 1 Recombinant plastid

DNA source

The PCV2-Cap (d41) fragment (shown as SEQ ID NO. 1) used in this case (Accession number: AY885225, 576 bp), Vlf-1 fragment (as shown in SEQ ID NO. 2) (Accession number: NC_001623) a partial fragment of .1, 1140 bp), a burst sequence fragment (shown as SEQ ID NO. 3), a Kozak sequence-GP64SP-His6 fragment (shown as SEQ ID NO. 4), a GP64 gene fragment ( GP64TMCTD (shown as SEQ ID NO. 5) (Accession number: partial fragment of NC_001623.1, 222 bp) was entrusted to Invitrogen for whole-gene synthesis, while codon modification was used as a sequence suitable for insect cell expression. BSms1 and BSms2 complete fragment amplification by primer pair PCR; the PCV2-Cap (d41) fragment refers to Capsid protein deletion of porcine circovirus type 2 (PCV2). The amino acid, more specifically, the PCV2-Cap (d41) fragment is used as a performance protein molecule which expresses the cardinal expression vector.

2. BSms1 polymerase chain reaction

1 μL (μl) [concentration 10 μmol concentration (μM)] P10 BSms1 F1 primer (as shown in SEQ ID No: 6), 1 μl (10 μM) P10 BSms1 R1 primer (eg SEQ ID No: 7), 2.5 μl 10X pfx buffer [50 millimolar (mM) Mg ⁺ ], 2.5 μl 10X enhancer, 2 μl dNTP (2.5 mM), primer pair, 0.5 μl pfx DNA polymerase, and finally deionized water To a total volume of 25 μl, put it into a PCR machine for reaction. The setting conditions were as follows: denaturation at 95 ° C for 3 minutes, followed by 95 ° C for 30 seconds, 55 ° C for "annealing" for 40 seconds, and 72 ° C for extension for 15 seconds. A total of 35 cycles were performed; finally, reaction was carried out at 72 ° C for 5 minutes to obtain BSms1 (as shown in SEQ ID No: 8).

3. BSms2 polymerase chain reaction

1 μl (10 μM) P10 BSms2 F1 primer (as shown in SEQ ID No: 9), 1 μl (10 μM) P10 BSms2 R1 primer (as shown in SEQ ID No: 10), 2.5 μl (50 mM Mg ⁺ ) 10X pfx buffer, 2.5 μl (2.5 mM) 10X enhancer, 2 μl dNTP, primer pair, 0.5 μl pfx DNA polymerase, finally added deionized water to a total volume of 25 μl, and placed in a PCR machine for reaction. The setting conditions were as follows: denaturation at 95 ° C for 3 minutes, followed by 95 ° C for 30 seconds, 55 ° C for 40 seconds, 72 ° C for 15 seconds for a total of 35 cycles; finally 72 ° C was reacted for 5 minutes to obtain BSms2 (as shown in SEQ ID No: 11).

4. PCR DNA identification and sequencing

After the completion of the PCR, the amplified DNA was subjected to DNA electrophoresis in 1X TAE buffer with SYBR 1% agarose gel, and the results were analyzed by UV light, and the sequence was confirmed to be correct after sequencing. confirm. As shown in Fig. 1, M denotes a DNA marker (Bio-100 bp Ladder); BSms1 denotes a BSms1 gene fragment amplified by Overlapping PCR; BSms2 denotes a BSms2 gene fragment amplified by Overlapping PCR, and the result shows that the product conforms to an expected fragment size of about 115 bp. .

5. DNA purification and conjugation reaction

The plastid and PCR DNA were cut with a restriction enzyme in a 37 ° C water bath for 1 hour, and the DNA of the reaction was analyzed by 1.5% agarose gel to determine the size of the insert and the vector. The DNA was recovered by purification using a Clean/Gel Extraction Kit. The target size DNA was cut out from the colloid, and the cut strip was added to Binding buffer [100 mg (mg): 100 μl], and placed in a 55 ° C water bath for the sol. After the colloid was dissolved, the liquid was added to the spin column. Centrifuge at 13,000 rpm for 30 seconds, remove the supernatant, add 700 μl of Wash buffer, centrifuge at 13,000 rpm for 30 seconds, and remove the supernatant. The spin column was then idling for 1 minute at 13,000 rpm in order to evaporate the alcohol. Add 40 μl of deionized water (D3W) to the spin column for 2 minutes, centrifuge at 13,000 rpm for 2 minutes, and the supernatant is the purified DNA. Calculate the molar ratio (insert: vector ratio 1:3), add an equal amount of T4 ligase in a 22 ° C water bath for 15 minutes.

6. Transformation (transformation)

In this case, the DH5α strain was used as a transformation. First, DH5α was taken out from -80 °C, placed on ice to slow down the freezing. After half of the dissolution, the ligation product was added and mixed, and placed on ice for 30 minutes to allow the DNA to adhere to the cell wall of the bacteria and maintain the cells. At 4 ° C, the heat shock was placed at 42 ° C for two minutes, the DNA was sent to the cells, and finally placed on ice for 10 minutes to heal the pores on the membrane. 100 μl of the bacterial solution was applied to an LB agar dish containing an antibiotic [containing 50 μg/ml ampicillin], and cultured at 37 ° C for 16 hours.

7. Extraction of recombinant plastid DNA

The white colonies after the transformation were cultured in a liquid state of 10 ml LB broth (containing 50 μg/ml ampicillin) at 37 ° C for 250 hours at rpm for 14 hours to 16 hours. Then take the bacteria solution at 3000 rpm for 10 minutes. After centrifugation, remove the supernatant. Use the Plasmid Miniprep Kit for plastid extraction. First add R3 buffer 200 μl to suspend the bacteria, then add L7 buffer 200 μl and gently mix it up and down. Egg-flower soup, and let stand for 5 minutes to 10 minutes to break the bacteria, and finally add 200 μl of N4 buffer, gently mix up and down, and simmer for 10 minutes at 13000 rpm. Take the supernatant to the spin column 13000 rpm for 1 minute (Labnet), remove the supernatant, add 500 μl of W10 buffer, centrifuge 13000 rpm for 30 seconds, discard the supernatant, add 600 μl of W9 buffer, centrifuge 13000 rpm for 30 seconds, then idling The alcohol was evaporated to dryness at 13,000 rpm for 10 minutes. Finally, the Spin column was placed in a 1.5 ml heart tube and added to 50 μl of deionized water (D3W) for 2 minutes at room temperature, and centrifuged at 13,000 rpm for 2 minutes. The collected liquid was reconstituted. body.

Preparation 2 Construction of a recombinant baculovirus vector

1. pFastBac ^TM Dual carrier

Vector pFastBac ^TM Dual case using Invitrogen's development, as shown in FIG. 2A, comprises two expression cassettes (expression cassette), wherein a sequence of p10 expression cassette promoter, multiple restriction sites (multiple cloning site, MCS The end-stop crypto fragment is HSV tk pA; the other expression cassette is the polyhedrin promoter (Pph) (sequence as shown in SEQ ID No: 12), the MCS, and the terminal stop chromosomal fragment is SV40 pA. Table 1 below lists the characteristics of each segment and its uses. Table 1. Characteristics of each section of the pFastBac ^TM Dual carrier and its use <TABLE border="1"borderColor="#000000"width="85%"><TBODY><tr><td>Features</td><Td> Properties and Uses</td></tr><tr><td> Polyhedrin Promoter (Pph) </td><td> Increases the expression of recombinant proteins in insect cells. </td></tr><tr><td> Multiple cloning site </td><td> allows for restriction enzyme entrapment into the sequence of interest. </td></tr><tr><td> SV40 pA </td><td> permits efficient transcription termination and polyadenylation of mRNA. </td></tr><tr><td> Tn7L and Tn7R </td><td> Miniature Tn7 allows site-specific transposition of the gene of interest to baculovirus In the genome. </td></tr><tr><td> f1 origin (f1 origin) </td><td> favors the production of single-stranded DNA products. </td></tr><tr><td> Anti-ampicillin resistance gene </td><td> allows screening of plastids in E. coli. </td></tr><tr><td> pUC origin </td><td> permits the replication and maintenance of high fractions of plastids in the E. coli gene. </td></tr><tr><td> gentamicin resistance gene </td><td> allows selection of recombinant rods in E. coli DH10Bac^TM Genome. </td></tr><tr><td> HSV tk pA </td><td> allows efficient transcription termination and polyadenylation of mRNA. </td></tr><tr><td> Multiple selection sites (P_p10) </td><td> allow selection of restriction enzymes into the sequence of interest. </td></tr><tr><td> The p10 promoter</td><td> allows efficient expression of recombinant proteins in insect cells. </td></tr></TBODY></TABLE>

2. Construct pBV1 performance vector

(1) The vector pFastBac ^TM Dual with BamHI and HindIII restriction enzyme cleavage at 37 ° C 1 h, the product to Gel / PCR DNA fragments extraction kit from DNA purified as colloidal Vector; GP64TMCTD as insert. Follow the reaction by joining, transition effects and screening recombinant plasmid was purified, to complete the pFastBac ^TM Dual-GP64TMCTD vector.

(2) The above pFastBac ^TM Dual-GP64TMCTD vector to Bstz17I and NotI restriction enzymes at 37 ° C 1 h, the product to Gel / PCR DNA fragments extraction kit DNA was purified from the colloid as Vector, the PH-BS2GP64SP construct to the carrier . Follow the reaction by joining, transition effects and screening recombinant plasmid was purified, to complete the vector pFastBac ^TM Dual-GP64.

(3) the pFastBac ^TM Dual-GP64 vector for NotI and ApaI restriction enzymes at 37 ° C 1 h, the product to Gel / PCR DNA fragments extraction kit DNA was purified from the colloid as Vector; PCV2-Cap (d41) as an insert . Follow the reaction by joining, transition effects and screening recombinant plasmid was purified, to complete the pFastBac ^TM Dual-GP64Cap (d41) vector.

(4) the pFastBac ^TM Dual-GP64Cap (d41) vector as a HindIII restriction enzyme cleavage at 37 ° C 1 h, the product to Gel / PCR DNA fragments extraction kit DNA was purified from the colloid as Vector; PH-EGFP-SV40pA as an insert . Subsequently, the ligation reaction, transformation, and recombinant plastid screening and purification were performed to complete the pBV1 expression vector shown in Fig. 3A, wherein an important regulatory fragment of pBV1 was constructed at the upstream of the transcription initiation point (+1) as shown in Fig. 3B. BS2 is about -10 bp to -94 bp; ATG in the Kozak sequence is the transcription initiation point (+1), and DNA polymerase expresses GP64SP, PCV2-Cap (d41) and other proteins downstream. Table 2 below lists the sequence start and end points and fragment length for each region. Table 2, pBV1 carrier information <TABLE border="1"borderColor="#000000"width="85%"><TBODY><tr><td>name</td><td> sequence start point</td><Td> sequence end point</td><td> fragment length</td></tr><tr><td> Ampicillin </td><td> 689 </td><td> 1549 </td><td > 861 </td></tr><tr><td> His6 </td><td> 4829 </td><td> 4846 </td><td> 18 </td></tr><Tr><td> Cap(d41) </td><td> 4871 </td><td> 5446 </td><td> 576 </td></tr><tr><td> EGFP </ Td><td> 5931 </td><td> 6647 </td><td> 717 </td></tr><tr><td> GP64TMCTD </td><td> 5465 </td><Td> 5554 </td><td> 90 </td></tr><tr><td> Tn7R </td><td> 2611 </td><td> 2835 </td><td> 225 </td></tr><tr><td> Gentamicin </td><td> 2902 </td><td> 3435 </td><td> 534 </td></tr><tr><td> BS2 </td><td> 4619 </td><td> 4702 </td><td> 84 </td></tr><tr><td> Kozak </td><td> 4709 </td><td> 4717 </td><td> 9 </td></tr><tr><td> SV40 pA </td><td> 6648 </td><td> 6888 </td><td> 241 </td></tr><tr><td> Tn7L </td><td> 6917 </td><td> 7082 </td><td> 166 </td></tr><tr><td> SV40 pA </td><td> 5561 </td><td> 5801 </td><td> 241 </td></tr><tr><td> Pph </td><td> 4478 </td><td> 4606 </td><td> 129 </td></tr><tr><td> Pph </td><td> 5802 </td><td> 5930 </td><td> 129 </td></tr><tr><td> f1 origin </td><td > 102 </td><td> 557 </td><td> 456 </td></tr><tr><td> pUC ori </td><td> 1694 </td><td> 2367 </td><td> 674 </td></tr><tr><td> GP64SP </td><td> 4718 </td><td> 4828 </td><td> 111 </td ></tr><tr><td> source </td><td> 1 </td><td> 7164 </td><td> 7164 </td></tr></TBODY></ TABLE>

3. Construct pBV2 performance vector

After the previously obtained pBV1 expression vector was cleaved with SmaI and KpnI restriction enzyme at 37 ° C for 1 hour, the product was purified from the colloid as a Vector using a Gel/PCR DNA fragments extraction kit; Vlf-1 was used as an insert. Subsequent ligation, transformation and recombinant plastid screening and purification to complete the pBV2 expression vector shown in Figure 4A, wherein the important regulatory fragment of pBV2 is constructed upstream of the transcription initiation point (+1) as shown in Figure 4B. BS2 is about -10 bp to -94 bp; ATG in the Kozak sequence is the transcription initiation point (+1), and DNA polymerase expresses GP64SP, PCV2-Cap (d41) and other proteins downstream. Table 3 below lists the sequence start and end points and fragment length for each region. Table 3, pBV2 carrier information  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> Name</td><td> Start of sequence</td><td> End of sequence</ Td><td> fragment length</td></tr><tr><td> f1 origin </td><td> 102 </td><td> 557 </td><td> 456 </td ></tr><tr><td> Ampicillin </td><td> 689 </td><td> 1549 </td><td> 861 </td></tr><tr><td> pUC ori </td><td> 1694 </td><td> 2367 </td><td> 674 </td></tr><tr><td> Tn7R </td><td> 2611 < /td><td> 2835 </td><td> 225 </td></tr><tr><td> Gentamicin </td><td> 2902 </td><td> 3435 </td> <td> 534 </td></tr><tr><td> HSV tk pA </td><td> 3992 </td><td> 4274 </td><td> 283 </td>< /tr><tr><td> Vlf-1 </td><td> 4281 </td><td> 5417 </td><td> 1137 </td></tr><tr><td> P10 </td><td> 5433 </td><td> 5554 </td><td> 122 </td></tr><tr><td> Pph </td><td> 5555 </ Td><td> 5683 </td><td> 129 </td></tr><tr><td> BS2 </td><td> 5696 </td><td> 5779 </td>< Td> 84 </td></tr><tr><td> Kozak </td><td> 5786 </td><td> 5794 </td><td> 9 </td></tr> <tr><td> GP64SP </td><td> 5795 </td><td> 5905 </td><td> 111 </td></tr><tr><td> His6 </td><td> 5906 </td><td> 5923 </td><td> 18 </td></tr><tr><td> Cap(d41) </td><td> 5948 </td><td> 6523 </td><td> 576 </td></tr><tr><td> GP64TMCTD </td><td> 6542 </td><td> 6631 </ Td><td> 90 </td></tr><tr><td> SV40 pA </td><td> 6638 </td><td> 6878 </td><td> 241 </td> </tr><tr><td> Pph </td><td> 6879 </td><td> 7007 </td><td> 129 </td></tr><tr><td> EGFP </td><td> 7008 </td><td> 7724 </td><td> 717 </td></tr><tr><td> SV40 pA </td><td> 7725 </ Td><td> 7965 </td><td> 241 </td></tr><tr><td> Tn7L </td><td> 7994 </td><td> 8159 </td>< Td> 166 </td></tr><tr><td> source </td><td> 1 </td><td> 8241 </td><td> 8241 </td></tr> </TBODY></TABLE>

4. Construct pBV10 performance carrier

After the previously obtained pBV2 expression vector was cleaved with restriction enzymes NdeI and SpeI restriction enzyme at 37 ° C for 1 hour, the product was purified from the colloid as a Vector using a Gel/PCR DNA fragments extraction kit; BSms1 was used as an insert. Subsequent ligation, transformation, and recombinant plastid screening purification to complete the pBV10 expression vector shown in Figure 5A, wherein the important regulatory fragment of pBV10 is constructed upstream of the transcription initiation point (+1) as shown in Figure 5B. BS2 is about -10 bp to -94 bp; BSms1 is about -10 bp to -107 bp; ATG in the Kozak sequence is the transcription initiation point (+1), and DNA polymerase expresses GP64SP and PCV2-Cap in the downstream direction (d41). ) and other proteins. Table 4 below lists the sequence start and end points and fragment length for each region. Table 4, pBV10 carrier information  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> Name</td><td> Start of sequence</td><td> End of sequence</ Td><td> fragment length</td></tr><tr><td> f1 origin </td><td> 102 </td><td> 557 </td><td> 456 </td ></tr><tr><td> Ampicillin </td><td> 689 </td><td> 1549 </td><td> 861 </td></tr><tr><td> pUC ori </td><td> 1694 </td><td> 2367 </td><td> 674 </td></tr><tr><td> Tn7R </td><td> 2611 < /td><td> 2835 </td><td> 225 </td></tr><tr><td> Gentamicin </td><td> 2902 </td><td> 3435 </td> <td> 534 </td></tr><tr><td> HSV tk pA </td><td> 3992 </td><td> 4274 </td><td> 283 </td>< /tr><tr><td> Vlf-1 </td><td> 4281 </td><td> 5417 </td><td> 1137 </td></tr><tr><td> P10 </td><td> 5433 </td><td> 5554 </td><td> 122 </td></tr><tr><td> Pph </td><td> 5555 </ Td><td> 5683 </td><td> 129 </td></tr><tr><td> BS </td><td> 5696 </td><td> 5737 </td>< Td> 42 </td></tr><tr><td> ms1 </td><td> 5738 </td><td> 5792 </td><td> 55 </td></tr> <tr><td> Kozak </td><td> 5799 </td><td> 5807 </td><td> 9 </td></tr><tr><td> GP64S P </td><td> 5808 </td><td> 5918 </td><td> 111 </td></tr><tr><td> His6 </td><td> 5919 </ Td><td> 5936 </td><td> 18 </td></tr><tr><td> Cap(d41) </td><td> 5961 </td><td> 6536 </ Td><td> 576 </td></tr><tr><td> GP64TMCTD </td><td> 6555 </td><td> 6644 </td><td> 90 </td>< /tr><tr><td> SV40 pA </td><td> 6651 </td><td> 6891 </td><td> 241 </td></tr><tr><td> Pph </td><td> 6892 </td><td> 7020 </td><td> 129 </td></tr><tr><td> EGFP </td><td> 7021 </td ><td> 7737 </td><td> 717 </td></tr><tr><td> SV40 pA </td><td> 7738 </td><td> 7978 </td>< Td> 241 </td></tr><tr><td> Tn7L </td><td> 8007 </td><td> 8172 </td><td> 166 </td></tr> <tr><td> source </td><td> 1 </td><td> 8254 </td><td> 8254 </td></tr></TBODY></TABLE>

5. Construct pBV11 performance carrier

After the previously obtained pBV2 expression vector was cleaved with restriction enzyme NdeI and SpeI restriction enzyme at 37 ° C for 1 hour, the product was purified from the colloid as a Vector using a Gel/PCR DNA fragments extraction kit; BSms2 was used as an insert. Subsequently, the ligation reaction, transformation, and recombinant plastid screening and purification were performed to complete the pBV11 expression vector shown in Fig. 6A, wherein an important regulatory fragment of pBV11 was constructed at the upstream of the transcription initiation point (+1) as shown in Fig. 6B. BS2 is about -10 bp to -94 bp; BSms2 is about -10 bp to -107 bp; ATG in the Kozak sequence is the transcription initiation point (+1), and DNA polymerase expresses GP64SP and PCV2-Cap in the downstream direction (d41). ) and other proteins. Table 5 below lists the sequence start and end points and fragment length for each region. Table 5, pBV11 carrier information  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> Name</td><td> Start of sequence</td><td> End of sequence</ Td><td> fragment length</td></tr><tr><td> f1 origin </td><td> 102 </td><td> 557 </td><td> 456 </td ></tr><tr><td> Ampicillin </td><td> 689 </td><td> 1549 </td><td> 861 </td></tr><tr><td> pUC ori </td><td> 1694 </td><td> 2367 </td><td> 674 </td></tr><tr><td> Tn7R </td><td> 2611 < /td><td> 2835 </td><td> 225 </td></tr><tr><td> Gentamicin </td><td> 2902 </td><td> 3435 </td> <td> 534 </td></tr><tr><td> HSV tk pA </td><td> 3992 </td><td> 4274 </td><td> 283 </td>< /tr><tr><td> Vlf-1 </td><td> 4281 </td><td> 5417 </td><td> 1137 </td></tr><tr><td> P10 </td><td> 5433 </td><td> 5554 </td><td> 122 </td></tr><tr><td> Pph </td><td> 5555 </ Td><td> 5683 </td><td> 129 </td></tr><tr><td> BS </td><td> 5696 </td><td> 5737 </td>< Td> 42 </td></tr><tr><td> ms2 </td><td> 5738 </td><td> 5792 </td><td> 55 </td></tr> <tr><td> Kozak </td><td> 5799 </td><td> 5807 </td><td> 9 </td></tr><tr><td> GP64S P </td><td> 5808 </td><td> 5918 </td><td> 111 </td></tr><tr><td> His6 </td><td> 5919 </ Td><td> 5936 </td><td> 18 </td></tr><tr><td> Cap(d41) </td><td> 5961 </td><td> 6536 </ Td><td> 576 </td></tr><tr><td> GP64TMCTD </td><td> 6555 </td><td> 6644 </td><td> 90 </td>< /tr><tr><td> SV40 pA </td><td> 6651 </td><td> 6891 </td><td> 241 </td></tr><tr><td> Pph </td><td> 6892 </td><td> 7020 </td><td> 129 </td></tr><tr><td> EGFP </td><td> 7021 </td ><td> 7737 </td><td> 717 </td></tr><tr><td> SV40 pA </td><td> 7738 </td><td> 7978 </td>< Td> 241 </td></tr><tr><td> Tn7L </td><td> 8007 </td><td> 8172 </td><td> 166 </td></tr> <tr><td> source </td><td> 1 </td><td> 8254 </td><td> 8254 </td></tr></TBODY></TABLE>

Preparation 3 Sf-9 and BTI-TN-5B1-4 (Hi-5) cell culture

In this case, the insect cell strains were Sf-9 ( Spoodoptera fugiperda ) and Hi-5 (BTI-TN-5B1-4) cells. The culture method can be cultured in a 28 ° C incubator using Insect-XPRESSTM Protein-free Insect Cell Medium. Sf-9 cells are subcultured for about 3 days; Hi-5 cells grow faster and can be subcultured once every 2 to 3 days. Two kinds of culture methods: The attached type is cultured in the flask, and when the cells are subcultured, the cells are suspended by tapping, and the number of 3×10 ⁶ cells is taken to the new T-75 flask. The suspension was cultured in a 1000 mL conical flask and placed in a 200 mL medium for 100 rpm shaking at a cell concentration of 1×10 ⁶ cells/mL.

Preparation Example 4 Preparation of Recombinant Baculovirus

1. Construction of a recombinant baculovirus gene (bacmid)

This preparation example uses the bac-to-bac baculovirus expression system developed by Invitrogen to prepare recombinant baculovirus, which can rapidly screen and construct recombinant baculovirus. The main baculovirus bac-to-bac expression system using a competent cell (competent cell) (DH10Bac ^TM) for construction of recombinant baculoviruses, the gene of this strain comprising a body portion baculovirus vector (shuttle vector) and helper plasmid Then, the previously constructed pBV1 expression vector, pBV2 expression vector, pBV10 expression vector and pBV11 expression vector were respectively transferred into the bacteria for genetic recombination. Due to the above carrier system as pFastBac ^TM Dual carrier substrate, i.e. each vector itself contains a gene and have Tn7R Tn7L the jump point and Yao Bacmid containing mini-attTn7 gene sequences, each of said current vectors were fed microbial cells, helper plasmid the The transposase will be translated and the target gene will be recombined with Bacmid using this property. After successful recombination, the lacZ gene of the Bacmid gene will be destroyed, unable to produce β-galatosidase, and then 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X -gal) and isopropylthio-β-galactoside (IPTG) were screened for blue and white. After 3 days to 4 days, blue and white colonies were observed. Finally, white colonies were cultured in liquid form and recombinant bacmid was extracted. In this step, a viral vector Bacmid-BD (negative control group), Bacmid-BV1, Bacmid-BV2, Bacmid-BV10 and Bacmid-BV11 were obtained, respectively. Although the baculovirus gene Bacmid of the present invention has the Vlf-1 gene, the efficiency of the transformation is considered, and the recombinant baculovirus vectors pBV2, pBV10 and pBV11 are constructed to express Vlf-1, and the transcription initiation point is regulated. (+1) Upstream zone BS2, BSms1 and BSms2 segments.

2. Transformational role

The construction of recombinant Bacmid completion BV1, BV2 recombinant Bacmid, the BV10 and BV11 recombinant Bacmid recombinant Bacmid manner into respectively heat shock DH10 Bac ^TM (commercially available from Invitrogen), the same procedure described above for the transformation, recovery of competent cells cultured for 3 hours Up to 5 hours, 30 μl to 80 μl of the inoculum was evenly spread on LA plate containing three antibiotics (50 μg/ml Kanamycin, 7 μg/ml Gentamycin, 10 μg/ml Tetracycline), and IPTG (40 μg/ml) was added. And X-gal (100 μg/ml) was cultured for 2 days to 3 days, and then white colonies were selected for liquid culture.

3. Purification of genetically recombinant Bacmid

The colonies after the proliferation culture were extracted with a Plasmid Purification Mini Kit. First, centrifuge 10 mL of the bacterial solution at 13,000 rpm, remove the supernatant, add 300 μl of buffer P1 back to the lysing block, add 300 μl of buffer P2 and mix it upside down for 10 times, let stand for 7 minutes at room temperature, and finally add 300 μl of buffer. P3 was inverted upside down and mixed uniformly and centrifuged at 13,000 rpm for 10 minutes. The QIAGEN-tip 20 was set up by centrifugation and first added to the equilibration column with 1 mL Buffer QBT. After centrifugation, the supernatant was added to the equilibrated QIAGEN-tip20, then the membrane in the equilibrated column was washed twice with 2 mL Buffer QC, and the DNA in the membrane was extracted into a 1.5 mL centrifuge tube with 800 μl Buffer QF. Add 500 μl of isopropanol and mix well. Centrifuge at 13000 rpm for 30 minutes, wash with 1 mL of 70% wash buffer, centrifuge at 13,000 rpm for 10 minutes, then discard the supernatant, air dry the DNA in a sterile console, and finally take 20 μl. Bacmid DNA was re-dissolved in deionized sterile water (D3W), and analyzed by PCR and electrophoresis to confirm whether the extracted recombinant Bacmid was homologously recombined successfully.

4. Transfection

The cultured Sf-9 cells were subcultured to a 6-well plate, and cultured at 1 × 10 ⁶ cells/mL per well, and cultured at 28 ° C for 1 hour. 2 μg of the above recombinant bacmid was diluted in 200 μl of Insect-XPRESSTM Protein-free Insect Cell Medium, and then 4 μl of TransIT ^® -Insect Transfection Reagent was added, mixed and allowed to stand at room temperature for 30 minutes. Then, 800 μl of the medium was added and mixed, and added to a 6-well plate at 28 ° C for 16 hours, and the transfection reagent was removed every other day to replace the fresh Insect-XPRESSTM Protein-free Insect Cell Medium. After continuous culture for 3 days, the supernatants were aspirated to obtain viruses BV1, BV2, BV10 and BV11 (all of which were P1 viruses).

5. Proliferating gene recombinant baculovirus

The purpose of the proliferating virus is to increase the viral power price to facilitate subsequent experimental use and virus preservation. First, 3×10 ⁶ Sf-9 cells were seeded to T25 flask, and insect cells were infected with MOI 1 (multiplicity of infection) virus solution, and cultured in a 28 ° C incubator for 3 days. When the cells were fluorescent, they were collected. The supernatant was centrifuged at 3000 rpm for 10 minutes. The suspension was mainly used to separate the suspended Sf-9 cells, and the virus was propagated for 3 passages, after which the virus was stored at -80 ° C or temporarily stored at 4 ° C.

6. Viral power price determination

The method for measuring the viral power rate is determined by the amount of fluorescence emitted after infection, and the viral power price is determined by the endpoint dilution method. Take 15 μl of the preceding virus solution were added to ^{135 μl Insect-XPRESS ™ Protein-} free Insect Cell Medium broth, and do serial diluted 10 times, after the diluted virus solution, each tube dilution was added 1215 μl (1 × 10 ⁵ cells /mL) Sf-9 cells, mix well, take 100 μl into a 96-well plate, add 12 wells at different dilution rates, and finally place in a 28 ° C incubator, and observe the cells to produce fluorescence for about 7 to 10 days. The degree of fluorescence expression of each dilution ratio was counted, and the viral power price TCID _{50 was} calculated by the formula (Reed-Muench method), and multiplied by 0.69 coefficient to be converted into pfu (plaque forming units).

Example 1 Identification of pBV1 expression vector, pBV2 expression vector, pBV10 expression vector and pBV11 expression vector

The construction process of pBV1 expression vector is described in Preparation Example 2.2. The construction process of pBV2 expression vector is detailed in Preparation Example 2.3. The construction process of pBV10 expression vector is shown in Preparation Example 2.4. The construction process of pBV11 expression vector is detailed in Preparation Example 2.5. The pBV1 expression vector was cleaved with NotI and HindIII; the pBV2 expression vector was cleaved with SmaI and KpnI; the pBV10 expression vector was based on the pBV2 expression vector, and the BS was replaced with BSms1, so the region fragment was identified by NdeI and SpeI cleavage. Yes, but because the fragment is too small, it is difficult to observe the target fragment, so it is detected by PCR. The pBV11 expression vector is based on the pBV2 expression vector, and the BS is replaced with BSms2. Therefore, the region fragment is identified by NdeI and SpeI. Since the segment is too small, it is difficult to observe the target segment, so it is detected by PCR. As shown in Figure 7A, after cleavage of the pBV1 expression vector, the GP64Cap (d41) gene fragment conforms to the expected size of 885 bp, and the PphEGFPSV40 pA gene fragment conforms to the expected size of 1176 bp; after the pBV2 expression vector is cleaved, the Vlf-1 promoter gene fragment meets The expected size is approximately 1140 bp. As shown in Fig. 7B, the pBV10 expression vector was detected by PCR, and showed that the expected size was 115 bp, and the pBV11 expression vector was detected by PCR, which showed that the expected size was 115 bp. As shown in Figure 7C, the nucleotides blast alignment of the NCBI website showed that the BSmsl sequence of the pBV10 expression vector (i.e., Sbjct shown in Figure 7C) was consistent with the Query sequence. As shown in Figure 7D, the nucleotides blast alignment of the NCBI website showed that the BSms2 sequence of the pBV11 expression vector (i.e., Subjct shown in Figure 7D) was consistent with the Query sequence.

Example 2 Identification of viruses BV1, BV2, BV10 and BV11

The recombinant baculovirus vectors Bacmid-BD, Bacmid-BV1, Bacmid-BV2, Bacmid-BV10 and Bacmid-BV11 were transfected into Sf-9 insect cells (see Preparation Example 4), and fired 3 days after transfection. According to light microscopy, since the EGFP gene is constructed on the vector, if there is a fluorescent expression, the vector is successfully sent into the cell to start producing a recombinant virus. The results of Fig. 8 show that Sf-9 insect cells all emit green fluorescence, so it can be determined that the viral vectors Bacmid-BD, Bacmid-BV1, Bacmid-BV2, Bacmid-BV10 and Bacmid-BV11 transfected insect cells can produce recombination rods. Virus. Since the baculovirus used in the present invention is AcMNPV, the assembled virus leaves the cells in a budding manner, and thus the supernatant contains the recombinant baculovirus. The recombinant baculovirus was named as virus BD, virus BV1, virus BV2, virus BV10 and virus BV11, respectively.

Example 3 Protein Representation Efficiency of Recombinant Baculovirus

The Hi-5 cells were cultured in a 250 mL conical flask (see Preparation Example 3), and the cell density was 10 ⁶ cells/mL, volume 25 mL, 28 ° C, and 100 rpm shaking culture. Hi-5 cells were infected with virus BV1, BV2, BV10, BV11 and MOI 0.1 for 3 days. The infected cells were subsequently centrifuged at 1500 rpm for 5 minutes to re-dissolve 1/10 fermentation volume of PBS. The 5X sample buffer dye was then diluted into 1X lysed cells and analyzed by protein electrophoresis and Western blotting.

For protein electrophoresis, the group included: Hi-5 and FastBacDual negative control group; BV1, BV2, BV10, BV11 experimental group, and E.coli showed the purified standard protein as a positive control group. The recombinant cell protein was quantified, mixed with 5X sample buffer dye and diluted to 1X, and the protein was completely denatured by a water bath at 100 ° C for 10 minutes. Then, electrophoresis was carried out by 10% SDS-PAGE, and 1X running buffer was injected. The upper layer of glue was run at 60 V for 30 minutes, and the lower layer of glue was run at 100 V for 90 minutes. After the colloid is run, the strip can be cut to analyze the protein by Western blotting.

The methanol-treated nitrocellulose membrane (NC membrane) membrane was coated on the SDS-PAGE colloid through a SDS-PAGE colloid. (The membrane must be immersed in the transfer buffer for 10 minutes before use.) When the above steps are completed, set the power supply to 400 mA for 45 minutes. The well-coated membrane was placed in a blocking buffer (containing 5% skim milk) and shaken at room temperature for 1 hour to reduce non-specific antibody binding. Subsequent addition of anti-β actin monoclonal antibody dilution ratio was diluted to 10000X, while anti-His6 monoclonal antibody dilution ratio was 5000X, shaking at room temperature for 1 hour to 2 hours. After the antibody was bound, it was washed 3 times with wash buffer (0.3% Tween 20 in PBS buffer) for 20 minutes each time, and the non-specific bond was washed away. Then take the diluted 5000X antibody (goat-anti-mouse (H+L) conjugate HRP, shake it at room temperature for 50 minutes, then wash it with appropriate amount of wash buffer 3 times for 20 minutes each time. (Anti-His6 monoclonal antibody has been To calibrate the HRP enzyme, no additional secondary antibody is required.

The NC membrane containing the protein was added to an enhanced chemiluminescence (ECL) solution (purchased from Amersham Pharmacia Biotech, New Territories), and divided into two tubes, A and B, each of which was mixed with A and B, and then covered with 200 μl. After reacting for an appropriate period of time on the NC membrane, the negative film was placed on the NC film, the image was presented on the negative film, and finally developed with a developing fixer.

Finally, the results were quantified by software Photo-CaptMW, and the data of each group were compared with the positive control group to calculate the protein expression. The data comparison between different groups was mainly analyzed by t-test function. The calculated P value was used as the statistical basis. If P<0.05, there was a significant difference; P>0.05 was the data, there was no significant difference. Determine the validity of different data. The data obtained by the Western blot method are divided by the β-actin data as a basis for quantification.

As a result, as shown in FIG. 9A and FIG. 9B, the four genetic recombinant baculoviruses have stable protein expression ability. The expected size Cap (d41) protein was detected by His6 monoclonal antibody at 28 kDa and 33 kDa, including Cap(d41)-gp64(TM-CTD) and Cap(d41)-gp64(SP-TM-, respectively. CTD) two types. Compared with BV1, the extra performance helps to regulate the expression of PCV2-Cap(d41) protein, and there are distinct differences in the amount of expression with different fragments such as BS, BSms1 and BSms2 at the 3' end of the promoter. Specifically, a specific region within 55 bp of the 5' end of the p10 promoter was modified (mutation of the AT sequence into GC) and then constructed downstream of the Pph promoter, wherein BV11 was optimally expressed. A standardized chart by software quantitative analysis showed that the Cap(d41) protein exhibited a significant 1.3-fold increase in BV11 performance compared to the BV1 performance.

Example 4 Analysis of protein yield in a fermenter mode

Hi-5 cells were cultured in a 5 L fermentation tank at a cell concentration of 10 ⁶ cells/mL, 80 rpm, DO maintained at 50%, pH 6.1, defoamer Pluronic ^® F-127 100 ppm, MOI 0.01, actual fermentation volume 1000 mL. Virus BV11 was infected for 3 days (Table 6 below). As a result, as shown in Fig. 10A, the survival rate of the cells was greatly decreased after 24 hours of infection, and was only about 50% at 72 hours of infection. As shown in Fig. 10B, the total number of cells was increased by about 40% compared with 0 hours. Table 6, 5L fermentation tank transport mode <TABLE border="1"borderColor="#000000"width="85%"><TBODY><tr><td> Recombinant baculovirus</td><td> infection Complex (MOI) </td><td> Infection days </td><td> Cell density (cells/mL) </td><td> revolutions per minute (RPM) </td><Td> Fermentation volume (mL) </td><td> Oxygen content</td><td> pH value </td><td> Defoamer concentration (ppm) </td></ Tr><tr><td> BV11 </td><td> 0.01 </td><td> 3 </td><td>10⁶</td><td> 80 </td><td> 1000 </td><td> 50% </td><td> 6.1 </td><td> 100 </td></tr></TBODY></TABLE>

Subsequent partial cell volume was performed by Western blotting method, and a group of purified proteins was added as a control, and a serial dilution was performed to linearize the bands on the electrophoresis patterns of 1 μg, 2 μg, 4 μg, 8 μg, and 16 μg. The function is derived, and finally the data of the target protein PCV2-Cap (d41) is substituted into the function to obtain the data. The results of Table 7 and Figure 11 below show that the total amount of BV11 after quantification is about 138 mg. Table 7. Quantitative analysis of PCV2-Cap (d41) protein  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> Experimental group</td><td> Hi-5 </td><td> BD </td><td> BV11 </td></tr><tr><td> Cap Protein (mg) </td><td> NA </td><td> NA </td><td> 138 </td></tr><tr><td> number of cells</td><td> 5x10⁷</td><td> 5x10⁷< /td><td> 5x10⁷</td></tr></TBODY></TABLE>NA means no detection.  

Figure 1 is a fragment of the BSms1 and BSms2 genes amplified by Overlapping PCR of the present invention; wherein M represents a DNA marker, and the fragment size of the BSms1 and BSms2 gene fragments is about 115 bp, respectively. FIG 2 is a schematic diagram of the vector pFastBac ^TM Dual used in the present invention. Figure 3A is a schematic illustration of the pBV1 expression vector of the present invention. Figure 3B is an important regulatory fragment of the pBV1 expression vector of the present invention, constructed at about -10 bp to -94 bp in the upstream position of the transcription initiation site (+1); ATG in the Kozak sequence is transcription At the starting point (+1), DNA polymerase expresses a schematic diagram of proteins such as GP64SP and PCV2-Cap (d41) in the downstream direction. Figure 4A is a schematic illustration of the pBV2 expression vector of the present invention. Figure 4B is an important regulatory fragment of the pBV2 expression vector of the present invention, constructed at about -10 bp to -94 bp in the upstream position of the transcription initiation point (+1); ATG in the Kozak sequence is the transcription initiation point (+ 1) DNA polymerase expresses a schematic diagram of proteins such as GP64SP and PCV2-Cap (d41) in the downstream direction. Figure 5A is a schematic illustration of the pBV10 expression vector of the present invention. 5B is an important regulatory fragment of the pBV10 expression vector of the present invention constructed at about -10 bp to -94 bp in the upstream position of the transcription initiation point (+1) BS2; about -10 bp to -107 bp in the BSms1; within the Kozak sequence The ATG is the transcription initiation point (+1), and the DNA polymerase expresses a schematic diagram of proteins such as GP64SP and PCV2-Cap (d41) in the downstream direction. Figure 6A is a schematic illustration of the pBV11 expression vector of the present invention. Figure 6B is an important regulatory fragment of the pBV11 expression vector of the present invention, constructed at about -10 bp to -94 bp in the upstream position of the transcription initiation site (+1); about -10 bp to -107 bp in BSms2; Kozak sequence The ATG inside is the transcription initiation point (+1), and the DNA polymerase expresses the GP64SP, PCV2-Cap (d41) and other proteins in the downstream direction. 7A is an electrophoresis pattern of the pBV1 expression vector and the pBV2 expression vector of the present invention, wherein the GP64Cap (d41) gene fragment meets the expected size of 885 bp, and the PphEGFPSV40 pA gene fragment conforms to the expected size of 1176 bp; the pBV2 expression vector is After cleavage, the Vlf-1 promoter gene fragment was approximately 1140 bp in size. 7B is an electrophoresis pattern of the pBV10 expression vector and the pBV11 expression vector of the present invention, which are detected by PCR, wherein the size of the pBV10 expression vector is 115 bp, and the size of the pBV11 expression vector is 115 bp. Figure 7C is a graph showing the alignment of the BSms1 sequence (Sbjct) and the sequencing result (Query) sequence of the pBV10 expression vector by the NCBI website (nucleotide blast). Figure 7D is a graph showing the alignment of the BSms2 sequence (Sbjct) and the sequencing result (Query) sequence of the pBV11 expression vector by the NCBI website (nucleotide blast). FIG 8 is a baculovirus vector Bacmid-BD (BD as pFastBac ^TM Dual abbreviation) of the present invention, Bacmid-BV1, Bacmid-BV2 , Bacmid-BV10 Bacmid-BV11 and transfected into Sf-9 insect cells three days After the visible light and the fluorescence map, the layer scale is 200 μm. 9A is the BD of the baculovirus of the present invention, BV1, BV2, BV10 and BV11 MOI0.1 infection in Hi-5 cells ⁽¹⁰⁶ cells total) exhibited the Cap (d41) comparing the electrophoretic protein exhibits FIG efficiency variance ;mock indicates that the negative control group has no virus. Figure 9B is a histogram of the quantitation of Figure 9A in software. Fig. 10A is a line graph showing the survival rate of the virus BV11 of the present invention in a fermentation tank for trial production, wherein the survival rate of Hi-5 cells after 24 hours of infection is drastically decreased, and continues to about 50% for 72 hours. Fig. 10B is a line graph showing the total number of cells in the fermentation tank of the virus BV11 of the present invention, wherein the total number of cells showed a small increase, and increased about 40% after 72 hours. Fig. 11 is a bar graph showing the protein expression of the virus BV11 of the present invention in a fermentation tank for trial production, wherein the 1 L fermentation tank can produce 138 mg of PCV2-Cap (d41) antigen protein.

<110> Hundred Health Technology Co., Ltd.

<120> For enhancing protein performance, calories, vectors, viruses and vaccines

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 576

<212> DNA

<213> Porcine circovirus 2 (Porcine circovirus 2)

<400> 1

<210> 2

<211> 1146 <212> DNA <213> Autographa californica moth nuclear polyhedrosis virus (Autographa californica nucleopolyhedrovirus) <400> 2 accatggtca acggattcaa cgtccgcaac gagaacaact tcaactcctg gaagatcaag 60 atccagtctg cccctcgctt cgaatctgtg ttcgacctgg ccaccgacag gcagaggtgc 120 acccccgacg aagtcaagaa caactctctg tggtctaagt acatgttccc caagcccttc 180 gcccccacca ccctgaagtc ttacaagtct cgcttcatca agatcgtcta ctgctctgtc 240 gacgacgtcc acctcgagga catgtcttac tccctggaca aggaattcga ctccatcgaa 300 aaccagaccc tgctgatcga cccccaagaa ctgtgcaggc gcatgctgga actgcgctct 360 gtcaccaagg aaaccctgca gctgaccatc aacttctaca ccaacatgat gaacctgccc 420 gagtacaaga tccccaggat ggtcatgctg cccagggaca aggaactgaa gaacatccgc 480 gaaaaggaaa agaacctgat gctgaagaac gtcatcgaca ccatcctgaa cttcatcaac 540 gacaagatca agatgctgaa ctccgactac gtccacgaca ggggactgat ccgcggagcc 600 atcgtgttct gcatcatgct gggaaccgga Atgaggatca acgaagccag gcagctgtcc 660 gtcgacgacc tgaacgtcct gatcaagagg ggaaagctgc actctgacac catcaacctg 720 aagaggaaga ggtctaggaa caacaccct g aacaacatca agatgaagcc cctggaactg 780 gccagggaaa tctactctag gaaccccacc atcctgcaga tctctaagaa cacctctacc 840 cccttcaagg acttccgcag gctgctggaa gaatctggtg tcgaaatgga aaggcccagg 900 tctaacatga tcaggcacta cctgtcctcc aacctgtaca actctggtgt ccccctgcaa 960 aaggtcgcca agctgatgaa ccacgaatct tccgcctcta ccaagcacta cctgaacaag 1020 tacaacatcg gactggacga aacctcctcc gaggaagaaa acaacaacga cgacgacgac 1080 gcccagcaca accgcaactc ttccggatct agcggagaat ctctgctgta ctaccgcaac 1140 gaataa 1146 <210> 3 <211> 84 <212> DNA <213> Artificial sequence <220><223>5' non-coding (leading) sequence of two-pulse sequence <400> 3 ctgttttcgt aacagttttg taataaaaaa acctataaat atctgttttc gtaacagttt 60 tgtaataaaa Aaacctataa atat 84 <210> 4 <211> 138 <212> DNA <213> Artificial sequence <220><223> Optimized GP64SPHis ₆ gene <400> 4 accatggtcc tggtcaacca gtctcaccag ggattcaaca aggaacacac ctctaagatg 60 gtgtccgcca tcgtcctgta cgtcctgctg gccgctgccg cccactctgc Cttcgc Tgct 120 catcaccatc accaccac 138 <210> 5 <211> 90 <212> DNA <213> Autographa californica nucleopolyhedrovirus <400> 5 tctatgttcc acgtcgtcaa cttcgtcatc atcctgatcg tcatcctgtt cctctactgc 60 atgatccgca accgcaacag gcagtactaa 90 <210> 6 <211> 68 <212> DNA <213> Artificial sequence <220><223> Forward introduction (P10 BSms1 F1) <400> 6 agtccgcata tgctgttttc gtaacagttt tgtaataaaa aaacctataa atatatacgg 60 acctttaa 68 <210> 7 <211> 61 <212> DNA <213> Artificial sequence <220><223> Reverse primer (P10 BSms1 R1) <400> 7 actagtaata attcttattt aactatccgg atccgtgttg ggttgaatta aaggtccgta 60 t 61 <210> 8 <211> 97 <212> DNA <213> Artificial sequence <220><223> BSms1 sequence, mutation position ggatccgg <400> 8 ctgttttcgt aacagttttg taataaaaaa acctataaat atatacggac ctttaattca 60 acccaacacg gatccggata gttaaataag aattatt 97 <210> 9 <21 1> 68 <212> DNA <213> Artificial sequence <220><223> Forward introduction (P10 BSms2 F1) <400> 9 agtccgcata tgctgttttc gtaacagttt tgtaataaaa aaacctataa atatatacgg 60 acctttaa 68 <210> 10 <211> 61 <212> DNA <213> Artificial sequence <220><223> Reverse primer (P10 BSms2 R1) <400> 10 actagtaata attcttattt aactatccgg atccgcgccg ggttgaatta aaggtccgta 60 t 61 <210> 11 <211> 97 <212> DNA <213> Artificial sequence <220><223> BSms2 sequence, mutation position is ggcgcggatccgg <400> 11 ctgttttcgt aacagttttg taataaaaaa acctataaat atatacggac ctttaattca 60 acccggcgcg gatccggata gttaaataag aattatt 97 <210> 12 <211> 129 <212> DNA <213> Autographa californica nucleopolyhedrovirus <400> 12 atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc 60 gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca 120 tcgggcgcg 129

Claims (8)

A bidirectional promoter expression cassette for enhancing protein expression, the bidirectional promoter expression cassette comprising: a first performance cassette comprising: a first baculovirus promoter; at least one a burst sequence (BS); a baculovirus GP64 gene sequence; and a terminator comprising a Simian vacuolating virus 40 polyadenylation (SV40 pA); and a second a performance cassette, operably linked in reverse to the first performance cassette, and the second performance cassette sequentially includes: a second baculovirus promoter; a transcription factor, wherein the transcription The factor is very late factor-1 (VLF-1); and, a terminator, including herpes simplex virus thymidine kinase polyadenylation (HSV tk pA) Wherein the first baculovirus promoter comprises a polyhedron promoter (Pph); wherein the second baculovirus promoter comprises a P10 promoter; wherein the at least one pulse The number of columns is one; and the pulse sequence and the baculovirus GP64 gene sequence further comprise a first mutation sequence (mutant signal 1, ms1), the pulse sequence and the first mutation sequence (BSms1) as SEQ ID No :8, or the pulse sequence and the baculovirus GP64 gene sequence further comprise a second mutation sequence (mutant signal 2, ms2), the pulse sequence and the second mutant sequence (BSms2) are shown as SEQ ID No: 11. The bidirectional promoter as described in claim 1 exhibits a cassette, wherein the baculovirus GP64 gene sequence sequentially includes a signal peptide (SP), a transmembrane domain (TM), and a cytoplasmic domain (cytoplasmic domain, CTD). The bidirectional promoter as described in claim 2 exhibits a cassette, wherein the signal peptide (SP) of the baculovirus GP64 gene sequence and the transmembrane region (TM) further comprise a plurality of restriction sites (multiple cloning sites). , MCS). The bidirectional promoter as described in claim 2 exhibits a cassette, wherein the signal peptide (SP) of the baculovirus GP64 gene sequence and the transmembrane region (TM) further comprise a porcine circovirus type II structural coat protein (d41). ) [porcine circovirus 2-capsid protein d41, PCV2-Cap (d41)]. A performance vector comprising the bidirectional promoter expression cassette of any one of claims 1 to 4. A genetically recombinant baculovirus formed by transfection of an expression vector as described in claim 5 to insect cells. The recombinant baculovirus according to claim 6, wherein the species of the insect cell comprises Sf-9 (Spodoptera fugiperda) cells or Hi-5 (BTI-TN-5B1-4) cells. A use of the expression vector of claim 5 for enhancing protein expression by transfecting an expression vector into an insect cell.