人尿激酶原cDNA在昆虫杆状病毒真核表达系统中的高效表达

人尿激酶原(pro-urokinase,pro-UK)是一种新型溶栓剂,优于尿激酶,具有血纤维蛋白特异性。为了在昆虫杆状病毒表达系统中高效表达pro-UK,我们在pAc373基础上,插入野生型AcMNPV polyhedrin启动子区-7～1碱基序列,构建了一个高表达转移载体pAcYT。分别经三次克隆将pro-UK cDNA正向插入到转移载体pAc373或PAcYT的BarnHI-KpnI位点上。用LiPofectin将pAcyT-UKDNA或pAc373-UK DNA与AcMNPV DNA共转染到昆虫Sf9细胞中,空斑法筛出重组病毒阳性克隆株。高效表达结果是:1.ELISA法确定重组病毒Sf9细胞分泌表达产物pro-UK为96mg/L培养基上清,平板法测定溶圈活性为1600IU/mL培养基上清;2.亲和层析一步法纯化表达产物,回收率选70％,以上,比活约为60000IU/mg;3.纯化的pro-UK,无论是否经还原处理,其SDS-PAGE图谱均为相同的单一条带,MR 50000;4.Western blot与SDS-PAGE图谱吻合。     

Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the expressed-protein stability

Human pro-urokinase (pro-UK) gene was engineered for expression in mammalian cells. The stability of recombinant pro-UKs produced by two kinds of cells, Chinese hamster ovary (CHO) and human lymphoblastoid Namalwa KJM-1 cells, were compared. The pro-UK expressed in CHO cells in serum-free medium was degraded by cysteine endopeptidase secreted by CHO cells. This endopeptidase was inhibited by pchloromercuribenzonate (PCMB) and leupeptin more efficiently than by aprotinin. On the other hand, the pro-UK expressed in Namalwa KJM-1 cells was not degraded, resulting in the stable production of pro-UK at a rate of 2–3 g/10⁶ cells/day by use of a gene amplification method with dihydrofolate reductase (DHFR) in serum-free medium. Thus, Namalwa KJM-1 cells showed the desired characteristics as a host cell for the production of recombinant proteins. The stability of recombinant proteins produced in heterologous systems may vary depending on the host cells.Abbreviations ABTS 2,2-azino-di-(3-ethylbenzothiazoline) sulfonic acid diammonium salt - AMC 7-amino-4-methylcoumarin - CHO Chinese hamster ovary - DFP diisopropylfluorophosphate - DHFR dihydrofolate reductase - ELISA enzyme-linked immunosorbent assay - MCA 4-methylcoumaryl-7-amide - MTX methotrexate - NEAA non essential amino acid - NEM N-ethylmaleimide - PCMB p-chloromercuribenzonate - PMSF phenylmethanesulfonyl fluoride - pro-UK pro-urokinase     

Efficient expression of pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells using Moloney retroviral promoter

We have compared the level of expression of several enhancer/promoters in human lymphoblastoid Namalwa KJM-1 cells when fused to a common reporter gene. A cassette containing the pro-urokinase (pro-UK) coding sequence followed by the rabbit -globin and simian virus 40 (SV40) 3 nontranslated region was used for evaluation of the enhancer activity. Cells containing Moloney murine leukemia virus (Mo-MuLV) promoter had an average of 10–20 fold higher expression levels of pro-UK than those containing other promoters, such as SV40 early gene promoter, human cytomegalovirus (hCMV) major immediate-early gene promoter, Rous sarcoma virus (RSV) promoter and chicken -actin gene promoter. The expression level of pro-UK under the control of Mo-MuLV promoter was 2–3 g/10⁶ cells/day and was constant for more than 6 months. Furthermore, the production of a high producer clone, obtained by using dhfr gene coamplification, reached 30–40 g/10⁶ cells/day. Thus, Mo-MuLV promoter showed the desired characteristics for efficient expression of foreign genes in Namalwa KJM-1 cells.Abbreviations dhfr dihydrofolate reductase - G-CSF granulocyte colony-stimulating factor - hCMV human cytomegalovirus - LTR long terminal repeat - Mo-MuLV Moloney murine leukemia virus - MTX methotrexate - pro-UK pro-urokinase - RSV Rous sarcoma virus - SV40 simian virus 40 - T3 triiodo-thyronine - TRE thyroid-hormone responsive element     

Efficient production of recombinant protein in Spodoptera frugiperda/AcNPV system utilizing silkworm hemolymph

Silkworm hemolymph when added at 5% (v/v) to medium increased the production of recombinant -galactosidase in Spodoptera frugipera/ Autographa californica nuclear polyhedrosis virus (AcNPV) system by 4.5-fold. Silkworm hemolymph also increased the host cell longevity by two-fold. Viability of the host cell was maintained at a constant level for 6 days after baculovirus infection in the medium containing 5% silkworm hemolymph, while host cells began to die 3 days after infection in the medium without hemolymph.     

Synthesis of functional bovine opsin in insect cells under control of the baculovirus polyhedrin promoter

In vitro expression of cDNA encoding bovine opsin is accomplished using the baculovirus expression vector system. Full-length opsin was synthesized which was recognized by poly- and monoclonal antisera raised against bovine rhodopsin. Upon infection with a recombinant virus, 1×10⁶ insect cells produced up to 3 g opsin. Incubation of the in vitro synthesized opsin with 11-cis retinal produced a hydroxylamine-stable, photosensitive pigment.Abbreviations dpi days post infection - pfu plaque forming units - AcNPV Autographa californica nuclear polyhedrosis virus     

Cloning and expression of the HN gene from the velogenic viscerotropic Newcastle disease virus strain AF2240 in Sf9 insect cells

The haemagglutinin-neuraminidase (HN) gene ofNewcastle disease virus (NDV) strain AF2240, amplifiedfrom the viral genomic RNA (1.8 kb) was directionallycloned and inserted into a baculovirus expressionvector system. The recombinant glycoprotein expressedin Spodoptera frugiperda (Sf9) cellsshowed haemagglutinin (HA), neuraminidase (NA) andhemadsorption activities. HA activity was detected inboth extra- and intra-cellular recombinant HN(recHNAF2240) samples. In addition, both HA andhemadsorption activities were inhibited by polyclonalanti-NDV sera. Furthermore, significant expression ofthe recombinant protein was observed on the surface ofinfected cells. SDS-PAGE analysis revealed thepresence of visually distinguishable bands between the70 and 80 kDa in size that were absent in thewild-type samples. Western blot analysis showed thatthe distinct 63 kDa band and a 75 kDa bandcorresponded to the unglycosylated and glycosylated HNglycoprotein respectively as reported in anotherstudy. These observations indicated that the HNrecombinant protein was not only expressed on thesurface of the infected cells as well as with theviral coat protein, but also appears to be functional.     

Effects of recombinant baculovirus AcMNPV-BmK IT on the formation of early cables and nuclear polymerization of actin in Sf9 cells

Autographa californica nuclearpoly hedrosis virus (AcMNPV) is one of the most important baculoviridae. However, the application of AcMNPV as a biocontrol agent has been limited. Previously, we engineered Buthus martensii Karsch insect toxin (BmK IT) gene into the genome of AcMNPV. The bioassay data indicated that the recombinant baculovirus AcMNPV-BmK IT significantly enhanced the anti-insect efficacy of the virus. The actin cytoskeleton is the major component beneath the surface of eukaryotic cells. In this report, the effects of AcMNPV-BmK IT on the formation of early cables of actin and nuclear filamentous-actin (F-actin) were studied. The results indicated that these baculovirus induced rearrangement of the actin cytoskeleton of host cells during infection and actin might participate in the transportation of baculovirus from cytoplasm to the nuclei. AcMNPV-BmK IT delayed the formation of early cables of actin and nuclear F-actin and accelerated the clearance of actin in the nuclei.     

Hepatocyte-specific gene expression by baculovirus pseudotyped with vesicular stomatitis virus envelope glycoprotein.

We have developed the recombinant baculovirus pseudotyped with vesicular stomatitis virus (VSV) G protein. The VSV-G gene was under the control of the polyhedrin promoter so that it was expressed at high levels in infected insect cells but not in mammalian cells. The presence of VSV-G protein in purified baculovirus preparations was confirmed by Western analysis. This recombinant baculovirus also carried human AFP (alpha-fetoprotein) promoter for hepatocyte-specific gene expression. After an in vitro infection by a recombinant baculovirus carrying the luciferase gene under the control of human AFP promoter/enhancer (BacG-AFP-Luc(+)), the luciferase gene was expressed in AFP-producing Huh7, Hep3B, and HepG2 cell lines, but not in AFP-nonproducing cell lines. BacG-AFP-Luc(+) transduced with human hepatoma cells in vitro at an efficiency about fivefold greater than the recombinant baculovirus lacking VSV-G (the virus Bac-AFP-Luc(+)). The utilization of the AFP promoter/enhancer in a baculovirus vector could provide benefits in gene therapy applications.     

虎源流感病毒HA 基因的系统发生分析及其在杆状病毒系统中的表达

通过对虎源流感病毒A/ Tiger/ Harbin/01/ 2003 (H5N1)的HA 基因进行克隆与序列测定,证明该基因全长为1 731 bp,读码框由1 707个碱基组成,编码568 个氨基酸。对HA 基因的进化分析表明,该基因与H5 亚型流感病毒的HA 基因同源性最高,其HA 裂解位点由6 个碱性氨基酸插入序列(RRRKKR)组成,符合高致病性禽流感病毒的分子特征。将HA 基因克隆入杆状病毒转座载体质粒pFastBacⅠ,构建重组质粒pFastBac-HA;再将该重组质粒转化DH10 Bac 感受态细菌,在体内进行重组,并经三重抗性与蓝白斑筛选,得到杆状病毒重组质粒Bacmid-HA;将Bacmid-HA 转染sf9 细胞,获得重组杆状病毒。经Western-blotting 检测,HA 蛋白在重组杆状病毒中获得表达。用感染重组病毒的sf9 细胞免疫小鼠,2 次免疫后2 周可诱导小鼠产生1∶ 8 ～1∶ 16 的血凝抑制抗体,表明虎源流感病毒的HA 基因在重组杆状病毒系统中得到了正确表达。     

Gastrointestinal Delivery of Baculovirus Displaying Influenza Virus Hemagglutinin Protects Mice against Heterologous H5N1 Infection

The recent outbreaks of influenza A H5N1 virus in birds and humans have necessitated the development of potent H5N1 vaccines. In this study, we evaluated the protective potential of an immediate-early promoter-based baculovirus displaying hemagglutinin (BacHA) against highly pathogenic avian influenza (HPAI) H5N1 virus infection in a mouse model. Gastrointestinal delivery of BacHA significantly enhanced the systemic immune response in terms of HA-specific serum IgG and hemagglutination inhibition (HI) titers. In addition, BacHA vaccine was able to significantly enhance the mucosal IgA level. The inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant along with BacHA vaccine did not influence either the systemic or mucosal immunity. Interestingly, an inactivated form of BacHA was able to induce only a negligible level of immune responses compared to its live counterpart. Microneutralization assay also indicated that live BacHA vaccine was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, and clade 8.0) compared to the inactivated BacHA. Viral challenge studies showed that live BacHA was able to provide 100% protection against 5 50% mouse lethal doses (MLD₅₀) of homologous (clade 2.1) and heterologous (clade 1) H5N1. Moreover, histopathological examinations revealed that mice vaccinated with live BacHA had only minimal bronchitis in lungs and regained their body weight more rapidly postchallenge. Furthermore, immunohistochemistry results demonstrated that the live BacHA was able to transduce and express HA in the intestinal epithelial cells in vitro and in vivo. We have demonstrated that recombinant baculovirus with a white spot syndrome virus (WSSV) immediate-early promoter 1 (ie1) acted as a vector as well as a protein vaccine and will enable the rapid production of prepandemic and pandemic vaccines without any biosafety concerns.The recent outbreaks of H5N1 avian flu and the current pandemic situation with H1N1 swine-origin influenza A virus (S-OIV) are clear indications of the urgent need for effective vaccines against influenza A viruses (31). Preventive and therapeutic measures against influenza A viruses have received much interest and effort globally to combat the current pandemic and to prevent such a situation in the future. Currently used vaccines for influenza are administered mainly parenterally and include live attenuated reassortant viruses, conventional inactivated whole viral antigens, or split-virus vaccines. Although some of these vaccines have proven to be quite effective, the manufacturing of these vaccines involves several technical and safety issues (21). Furthermore, the production of currently available influenza vaccines often requires high-level biocontainment facilities, an additional hurdle that limits the advancement of present vaccines.Vaccines containing purified recombinant viral proteins have recently gained special attention due to their ease of production without any safety concerns (25). Recombinant hemagglutinin (rHA) subunit vaccines produced in baculovirus-insect cell expression systems have been extensively tested and evaluated in humans (29, 30). Baculovirus-derived rHA subunit vaccines administered parenterally are safe and immunogenic in animals and humans. Along with its success in recombinant protein vaccines, baculovirus surface display technology allows us to present large complex proteins on the baculovirus envelope in its native antigenic conformation, resulting in good stability and a longer half-life in the host (18, 14, 8).Along with a suitable antigen, the route of administration of the vaccine has a profound effect in controlling mucosally acquired infections such as influenza. Vaccination via the mucosal route stimulates both systemic and mucosal immune responses (16). Oral and intranasal vaccines are the two main options for mucosal administration. Intranasal vaccines would have a detrimental effect on persons with asthma, reactive airway disease, and other chronic pulmonary or cardiovascular disorders (4). Oral vaccines therefore seem to be the safest alternative (13). Moreover, there is evidence to prove the ability of oral vaccination to prevent infection of the lungs (23) and cause transcytosis of the molecule across the cells into the circulation (24).In this report, we describe the construction of recombinant baculovirus under control of the immediate-early promoter 1 (ie1) derived from the white spot syndrome virus (WSSV) genome, which enables the expression of hemagglutinin at the early stage of infection in insect cells, thereby enhancing the display of HA on the baculovirus envelope. Incorporation of more HA into the budding baculovirus particles would improve their efficacy as immunogens. We have studied the efficacy of WSSV ie1-based baculovirus displaying hemagglutinin (BacHA) as an oral vaccine in a mouse model of infection. We have also assessed its efficacy with recombinant cholera toxin B (rCTB) as a mucosal adjuvant. This strategy will enable rapid production of prepandemic vaccines with minimal infrastructure around the world, alleviating the need for high-biosafety facilities, risky inactivation of virulent viruses, and meticulous protein purification procedures.     

Expression of a foreign gene by cysteine proteinase null recombinant baculovirus

Baculovirus expression vector systems (BEVSs) are broadly used for producing foreign proteins in lepidopteran cells. Most commercial BEVSs are engineered to insert foreign genes into the polyhedrin (polh) locus. They lack the polh gene. These viruses cannot produce occlusion bodies and are inconvenient for per os inoculation of larvae. To avoid this, expression cassettes can be inserted in other parts of the virus genome. The preS2-S gene, coding for the recombinant middle surface antigen of the human hepatitis B virus (M-HBsAg), was expressed from the baculovirus construct rBmNPV-Δv-cath-M-HBsAg, inserting the foreign gene into the v-cath locus of Bombyx mori nucleopolyhedrovirus (BmNPV) so that v-cath was deleted and native polh was retained. Silkworm larvae were infected per os and M-HBsAg was observed to be abundantly produced till very late stages of infection. Infection of larvae with a mixture of the recombinant and wild-type baculoviruses was followed by degradation of the bulk of the produced M-HBsAg as early as 96 h after inoculation.     

Baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice

A recombinant baculovirus expressing the hemagglutinin gene of the influenza virus, A/PR/8/34 (H1N1), under the control of the chicken beta-actin promoter, was constructed. To determine the induction of protective immunity in vivo, mice were inoculated with the recombinant baculovirus by intramuscular, intradermal, i.p., and intranasal routes and then were challenged with a lethal dose of the influenza virus. Intramuscular or i.p. immunization with the recombinant baculovirus elicited higher titers of antihemagglutinin Ab than intradermal or intranasal administration. However, protection from a lethal challenge of the influenza virus was only achieved by intranasal immunization of the recombinant baculovirus. Surprisingly, sufficient protection from the lethal influenza challenge was also observed in mice inoculated intranasally with a wild-type baculovirus, as evaluated by reductions in the virus titer, inflammatory cytokine production, and pulmonary consolidations. These results indicate that intranasal inoculation with a wild-type baculovirus induces a strong innate immune response, which protects mice from a lethal challenge of influenza virus.     

Glycosylation of prourokinase produced by Pichia pastoris impairs enzymatic activity but not secretion

Both glycosylated and nonglycosylated forms of recombinant human prourokinase were produced to the level of 20 mg/L by yeast Pichia pastoris in BMMY medium after 2 days of culture. The expressed pro-UK was 98% secreted into the culture medium and easily purified by carboxymethyl cellulose chromatography. More than 99% of pro-UK in the culture medium was found in single-chain form. This was contradictory to a previous finding which found that glycosylation of pro-UK by yeast inhibited its secretion. The absence of glycosylation at Asn302 of pro-UK has no measurable effect on its secretion from the yeast cells. However, the nonglycosylated pro-UK was much less stable in the culture medium, probably due to proteolysis. Nonglycosylated pro-UK from yeast had a clot lysing activity comparable to that of Escherichia coli-derived or mammalian cell-derived recombinant pro-UK. By contrast, the glycosylated yeast pro-UK was less activatable by plasmin and had a lower enzymatic activity against plasminogen and a lower clot lysing activity than nonglycosylated pro-UK from yeast, while their amidolytic activity against S2444 was equivalent. It was concluded that glycosylation of pro-UK by yeast P. pastoris interferes with the catalytic site but not secretion of this protein.     

Electrophoretic analysis of glycoprotein glycans produced by lepidopteran insect cells infected with an immediate early recombinant baculovirus encoding mammalian β1,4-galactosyltransferase

Glycosylation, the most extensive co- and post-translational modification of eukaryotic cells, can significantly affect biological activity and is particularly important for recombinant glycoproteins in human therapeutic applications. The baculovirus-insect cell expression system is a popular tool for the expression of heterologous proteins and has an excellent record of producing high levels of biologically active eukaryotic proteins. Insect cells are capable of glycosylation, but their N-glycosylation pathway is truncated in comparison with the pathway of mammalian cells. A previous study demonstrated that an immediate early recombinant baculovirus could be used to extend the insect cell N-glycosylation pathway by contributing bovine -1,4 galactosyltransferase (GalT) immediately after infection. Lectin blotting assays indicated that this ectopically expressed enzyme could transfer galactose to an N-linked glycan on a foreign glycoprotein expressed later in infection. In the current study, glycans were isolated from total Sf-9 cell glycoproteins after infection with the immediate early recombinant baculovirus encoding GalT, fluorescently conjugated and analyzed by electrophoresis in combination with exoglycosidase digestion. These direct analyses clearly demonstrated that Sf-9 cells infected with this recombinant baculovirus can synthesize galactosylated N-linked glycans.     

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

Background

Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene.

Methods

A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein.

Results

Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts.

Conclusions

In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

     

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N‐terminally His‐tagged NiV M protein in insect cells. A time‐course study demonstrated that the highest yield of recombinant M protein (400–500 μg) was expressed from infected cells 3 days after infection. A single‐step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme‐linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:171–177, 2016     

Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 × 10⁶ cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) cross-reacted with re-PNA. The subunit molecular weight (30kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.     

CHO工程细胞 (11G-S) 悬浮培养的无血清培养基的设计

以悬浮适应的表达重组尿激酶原 (Pro-urokinase,pro-UK) CHO工程细胞系11G-S为对象,采用Plackett-Burman实验设计及响应面分析法,设计支持CHO工程细胞 (11G-S) 悬浮生长的无血清培养基。以细胞密度为评价指标,在单因素实验的基础上采用Plackett-Burman实验设计对影响细胞生长的培养基添加成分进行考察,确定了3种对细胞生长明显促进作用的培养基添加成分：胰岛素、转铁蛋白及腐胺。继而利用响应面法分析了这3种添加成分的最佳水平范围,设计了一种适用于CHO工程细胞 (11G-S) 悬浮培养的无血清培养基SFM-CHO-S。11G-S细胞在SFM-CHO-S批次悬浮培养的细胞最大生长密度达到4.12×106 cells/mL,pro-UK的最大累积活性达到5 614 IU/mL,培养效果优于商品化的同类无血清培养基。     

甲型H1N1流感病毒HA基因在昆虫细胞中的截短表达

目的:利用Bac-to-Bac Baculovirus Expression System表达重组HA蛋白,Western blot及IFA方法鉴定其表达。方法:采用PCR方法扩增A/California/04/2009(H1N1)HA基因,将其克隆到pFastBacHT A载体上,重组质粒pFastBacHT-HA经双酶切及测序鉴定正确后,转化阳性重组载体进入E.coli DH10Bac感受态细胞中,通过Bluo-gal蓝白斑筛选、PCR鉴定获得重组转座子rBacmid-HA。从重组转座子中提取rBacmid-HA质粒DNA转染sf 9昆虫细胞,制备重组杆状病毒。重组杆状病毒感染sf 9细胞表达重组蛋白,Western blot及IFA鉴定重组蛋白表达情况。结论:成功构建了甲型H1N1流感病毒HA基因的昆虫杆状病毒表达载体,该表达载体转染昆虫细胞后制备的重组杆状病毒病毒滴度较高,重组杆状病毒表达的重组蛋白经Western blot 及IFA 鉴定后具有良好的免疫反应原性。     

牛λ3干扰素在家蚕杆状病毒表达系统中的表达及其抗病毒活性检测

牛λ3干扰素(BoIFN-λ3)是一种新型干扰素,可应用于牛传染性疾病的防治。在家蚕杆状病毒表达系统中可实现BoIFN-λ3的高效表达。首先在优化合成的BoIFNλ3基因起始密码子上游引入Kozak序列,将其克隆至转移载体pVL1393,获得pVL1393-BoIFN-λ3重组质粒。利用本实验室构建的家蚕杆状病毒表达系统,获得整合BoIFN-λ3基因的重组家蚕杆状病毒,将重组病毒感染五龄起蚕,在蚕血淋巴中得到表达产物BoIFN-λ3。采用微量细胞病变抑制法在MDBK/VSV*GFP 系统检测蚕体中表达BoIFN-λ3的效价可达（2.7±0.12）×105 U/mL,利用空斑筛选法筛选重组病毒,测得最高表达量的重组病毒表达的BoIFN-λ3的效价可达（8.1±0.52）×105 U/mL,表达量提高3倍。家蚕杆状病毒表达系统为优质高效的牛λ3干扰素产品的生产提供了一种新方法。