猪γ-干扰素在重组杆状病毒中的表达及其抗病毒活性的测定

研究利用Bac-To-Bac杆状病毒表达系统构建含有猪γ-干扰素(porcine interferon-γ,PoIFN-γ)完整开放阅读框的供体质粒pFastBacTM1-PoIFN-γ,转化DH10Bac感受态细胞获得重组穿梭质粒rBacmid-PoIFN-γ,转染sf9昆虫细胞救获表达PoIFN-γ的重组杆状病毒rBac-PoIFN-γ。以抗PoIFN-γ单克隆抗体为一抗进行Western blot、间接免疫荧光(IFA)及间接ELISA检测,结果表明PoIFN-γ在重组杆状病毒rBac-PoIFN-γ感染的昆虫细胞中获得正确表达。抗病毒活性试验显示,重组杆状病毒表达rPoIFN-γ能有效抑制水疱性口炎病毒(VSV)在猪肾细胞系(PK-15)的复制,rBac-PoIFN-γ感染昆虫细胞培养上清抗病毒活性为2×104抑制单位(IU)/mL,其抗病毒活性可以被鼠抗原核表达重组PoIFN-γ免疫血清有效阻断。结果表明rPoIFN-γ在重组杆状病毒rBac-PoIFN-γ在感染昆虫细胞获得有效表达,并具有高效抗病毒活性。     

重组杆状病毒表达牛β干扰素及其生物学活性研究

本研究构建了表达牛β干扰素的重组杆状病毒,感染sf9细胞后,分别采用免疫荧光和免疫印迹证实了重组BoIFN-β的表达存在于细胞内和培养上清中。采用表达绿色荧光蛋白的水疱性口炎病毒(VSV*GFP)检测分泌到细胞上清中重组BoIFN-β的抗病毒活性,可达到106.0AU/mL。同时重组BoIFN-β还可以激活鸡Mx启动子控制的萤光素酶报告基因的转录表达。综上,本研究采用杆状病毒表达系统,重组牛β干扰素以分泌形式高水平表达,且具有天然Ⅰ型干扰素的生物学活性。     

牛γ 干扰素的表达及其抗病毒活性测定

通过RT-PCR从经ConA刺激诱导的奶牛脾脏淋巴细胞总RNA中扩增出牛γ干扰素 (BoIFN-γ) cDNA,克隆到真核载体pVAX1中,测序结果显示pVAX1中的插入序列BoIFN-γ基因与已报道序列一致。用重组质粒pVAX1-BoIFN-γ转染COS-7细胞并进行间接免疫荧光试验鉴定,结果显示BoIFN-γ在COS-7细胞中得到成功表达。将BoIFN-γ基因克隆到原核表达质粒pET-30a(+)、pGEX-6p-1后,分别转化重组表达菌BL21(DE3)、BL21后,通过对表达条件的优化,SDS-P     

鸡γ-干扰素在杆状病毒系统中的表达

目的：获得具有生物学活性的重组鸡γ-干扰素（ChIFN-γ）。方法：应用RT-PCR方法扩增ChIFN-γ基因cDNA,将其克隆入杆状病毒表达系统的转移载体pFastBac1,构建重组质粒pFast-ChIFN-γ,通过位点特异性转座,将ChIFN-γ基因整合到Bacmid穿梭载体中,构建重组质粒pBac-ChIFN-γ并转染昆虫细胞Sf9,采用间接免疫荧光试验（IFA）、ELISA试验鉴定重组ChIFN-γ的表达,通过细胞病变抑制法检测重组蛋白的抗病毒活性。结果：IFA、ELISA与细胞病变抑制试验结果显示,重组ChIFN-γ在杆状病毒系统中获得表达,其抗病毒活性达5×103～1×104U/mL。结论：重组ChIFN-γ的获得为其开发应用提供了重要的生物材料。     

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。     

猪γ干扰素的表达及其抗病毒活性的安全检测

为了提高干扰素抗病毒活性测定的生物安全性,本研究使用含有GFP的复制缺陷型水疱性口炎病毒(VSV△G*G)为指示病毒,分别对经原核表达系统和杆状病毒表达系统表达的重组猪γ干扰素(PoIFN-γ)在MDBK细胞上进行抗病毒活性测定。结果显示:由杆状病毒表达的重组PoIFN-γ具有高度抗病毒活性,其抗病毒活性为105IU/mL,原核表达的重组PoIFN-γ纯化后经缓慢复性也会产生一定的抗病毒活性,其抗病毒活性为32IU/mL。该方法与利用表达GFP的重组水疱性口炎病毒(VSV*GFP)所检测的干扰素抗病毒活性结果完全一致,表明复制缺陷型病毒VSV△G*G可作为复制型重组病毒的替代品,使得干扰素抗病毒活性检测更加安全、准确。     

北极狐γ-干扰素cDNA的克隆、表达及活性测定

为研究狐γ-干扰素的生物学活性,应用反转录聚合酶链式反应(RT-PCR)从北极狐外周血淋巴细胞中扩增出γ-干扰素(VuIFN-γ)cDNA.序列分析表明VuIFN-γcDNA全长501 bp,编码23个氨基酸的信号肽和144个氨基酸的成熟肽蛋白,与已发表的银黑狐和犬IFN-γ核苷酸序列同源性为99.8%和99.4%;氨基酸同源性均为100%.应用原核表达系统高效表达北极狐γ-干扰素成熟肽蛋白,SDS-PAGE和Western blotting分析表达的融合蛋白分子量约为19 kD,以不溶性的包涵体形式存在.重组蛋白经纯化和复性,在Vero和MDCK细胞上可明显抑制VSV病毒的复制,并测出北极狐重组γ-干扰素的活性单位分别为1.0×106 u/mg和1.56×105 u/mg,为进一步开发基因工程狐干扰素奠定基础.     

CHO细胞表达重组猪干扰素-γ及其抗病毒活性

为利用中国仓鼠卵巢(Chinese hamster ovarian,CHO)细胞表达系统制备重组猪干扰素-γ(recombinant porcine interferon gamma,r Po IFN-γ),并分析其体外抗病毒活性,本研究首先构建r Po IFN-γ真核表达质粒pc DNA3.1-Po IFN-γ,转染悬浮培养的CHO细胞,进行上清分泌表达,利用亲和层析纯化目的蛋白,并进行SDS-PAGE和Western blotting鉴定;通过CCK-8实验分析r Po IFN-γ对细胞的毒性,用VSV/PK-15系统检测其抗病毒活性效价;最后分析r Po IFN-γ对塞内卡病毒A (Seneca virus A,SVA)的抗病毒活性及其对细胞内干扰素刺激基因(interferon-stimulated genes,ISGs)和细胞因子的诱导作用。结果显示,本研究利用CHO悬浮细胞表达系统成功制备了纯化的r Po IFN-γ,该r Po IFN-γ对细胞无毒性,VSV/PK-15系统检测其活性效价为5.59×107 U/mg;此外,r Po IFN-γ可诱导细胞内多种ISGs和细胞...     

重组鸡γ_干扰素在昆虫细胞中的高效表达

将鸡γ_干扰素(ChIFN_γ)基因克隆到载体pFASTBAC1中,构建转移载体pFASTBAC1_ChIFN_γ,然后转化DH10Bac感受态大肠杆菌,通过位点特异性转座,将ChIFN_γ基因整合到Bacmid穿梭载体中,构建表达质粒Bacmid_ChIFN_γ;通过脂质体将表达质粒转染Sf9昆虫细胞,用间接免疫荧光试验鉴定重组鸡γ_干扰素（rChIFN_γ）的表达;通过水泡性口炎病毒感染鸡胚成纤维细胞（CEF）的细胞病变抑制试验,检测rChIFN_γ的活性。研究结果表明,感染重组杆状病毒的Sf9细胞能高效表达rChIFN_γ,且当每个细胞的感染量为1个病毒时,细胞在感染96h后,rChIFN_γ基因表达产物的活性最高,达到10.6～10 7.2U/mL。 以rChIFN_γ进行对新城疫病毒(NDV) F48E8株、禽流感H5N1病毒（AIV_H5）和马立克氏病病毒 (MDV) GA株的抗病毒活性试验,发现rChIFN_γ对AIV_H5和MDV GA株病毒有明显的抑制其致细胞病变作用,但对NDV F48E8株病毒在体外不能抑制其致细胞病变作用,仅能在病毒滴度上表现抑制效果。     

重组鸡γ_干扰素在昆虫细胞中的高效表达

将鸡γ-干扰素(ChIFN-γ)基因克隆到载体pFASTBAC1中,构建转移载体pFASTBAC1-ChIFN-γ,然后转化DH10Bac感受态大肠杆菌,通过位点特异性转座,将ChIFN-γ基因整合到Bacmid穿梭载体中,构建表达质粒Bacmid-ChIFN-γ;通过脂质体将表达质粒转染Sf9昆虫细胞,用间接免疫荧光试验鉴定重组鸡γ-干扰素(rChIFN-γ)的表达;通过水泡性口炎病毒感染鸡胚成纤维细胞(CEF)的细胞病变抑制试验,检测rChIFN-γ的活性。研究结果表明,感染重组杆状病毒的Sf9细胞能高效表达rChIFN-γ,且当每个细胞的感染量为1个病毒时,细胞在感染96h后,rChIFN-γ基因表达产物的活性最高,达到106~107.2U/mL。以rChIFN-γ进行对新城疫病毒(NDV)F48E8株、禽流感H5N1病毒(AIV-H5)和马立克氏病病毒(MDV)GA株的抗病毒活性试验,发现rChIFN-γ对AIV-H5和MDV GA株病毒有明显的抑制其致细胞病变作用,但对NDVF48E8株病毒在体外不能抑制其致细胞病变作用,仅能在病毒滴度上表现抑制效果。     

Production of recombinant bovine lactoferrin N-lobe in insect cells and its antimicrobial activity

Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation.     

Cell lines used for the selection of recombinant baculovirus

Summary Four insect cell lines were used to isolate two recombinant baculoviruses which had theβ-galactosidase (β-gal) gene for colorimetric assay purposes. Plaque assays were performed using twoTrichoplusia ni cell lines: BTI-TN-5B1-4 and TN-368, and twoSpodoptera frugiperda cell lines: IPLB-SF-21AE and SF9. The number of plaques (occlusion positive and blueβ-gal⁺ recombinants) formed in theTrichoplusia cells was higher than in theSpodoptera cells. The appearance ofAutographa californica NPV polyhedra was also faster in theT. ni cell lines. The effect of cell passage on the plaque formation proved to be critical when two different passages of the SF9 cells were tested. The higher passage produced a lower viral titration. The size and time of appearance of the plaques was also different.     

Recombinant gamma interferon enhances natural killer cell activity similar to natural gamma interferon

The enhancement of human natural killing activity by recombinant human gamma interferon (IFNγ) and natural human IFNγ were similar over a wide concentration range. Enhancement of natural killing activity by both interferons was neutralizable by antibody to natural IFNγ, as well as by antibody to a synthetic peptide representing the first 20 N-terminal amino acids of IFNγ provide conclusive evidence that IFNγ is responsible for the enhanced natural killing activity seen in IFNγ preparations.     

Expression and one-step purification of bovine interleukin-21 (IL-21) in silkworms using a hybrid baculovirus expression system

A hybrid baculovirus, a hybrid of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of bovine interleukin-21 (IL-21) in silkworms. A recombinant hybrid baculovirus containing the full length of the cDNA of bovine interleukin-21 was constructed and used to infect silkworm larvae or silkmoth pupae. After the infection of the virus, bovine mature IL-21 was produced in the haemolymph or pupal cell lysates. A one-step purification of bovine mature IL-21 from haemolymph using a cation exchange column gave 0.5 mg. IL-21 from 30 ml haemolymph. The bovine IL-21 produced by silkworms strongly induced NK cell proliferation using a human NK cell-line, NK0, and enhanced the lymphokine activated killer (LAK) activity of bovine peripheral blood mononuclear cells.     

Genetic polymorphisms and antiviral activity in the bovine MX1 gene

Bovine MX1 cDNAs consisting of 2280 bp from 11 animals of five breeds and from a cultured cell line were sequenced and compared with previously reported data. Ten nucleotide substitutions were synonymous mutations, and a single nucleotide substitution at 458 resulted in an amino acid exchange of Ile (ATT) and Met (ATG). A 13-bp deletion-insertion mutation was also found in the 3'-UTR. Based on the nucleotide substitutions found in this study, bovine MX1 cDNA was classified into 11 genotypes. A phylogenetic tree of the 11 genotypes suggested that the genotypes observed in Brahman were a great genetic distance from other genotypes. An 18-bp deletion-insertion variation at position 171 was found to be the result of alternative splicing. The 18-bp deletion-insertion is located at the boundary between exon 3 and intron 3. Permanently transfected 3T3 cell lines expressing bovine MX1 mRNA were established to analyse the antiviral potential against VSVDeltaG*-G infection. Transfected cell clones expressing bovine MX1 mRNA showed a significantly smaller number of cells infected with VSVDeltaG*-G compared with the control cells. These results indicate that the bovine MX1 protein has potent antiviral activity.     

Expression of equine interleukin-18 by baculovirus expression system and its biologic activity

The equine interleukin-18 (IL-18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL-18, was constructed. The recombinant protein of the equine IL-18 was expressed by AcEIL-18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Insect cells infected with AcEIL-18 secreted a precursor IL-18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL-18 about 18 kDa was also confirmed without co-expression of caspase-1. Culture supernatant from AcEIL-18 infected cells showed a synergistic effect with recombinant human interleukin-12 for induction of interferon-gamma gene expression in equine peripheral mononuclear cells, indicating that the recombinant equine IL-18 expressed in this study also has biological activity without any treatment.     

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.     

Factors affecting the dissociation and aggregation of human interferon gamma

The biologically active form of interferon γ (IFN-γ) is a dimer consisting of two identical non-covalently bound polypeptide chains. We have studied spectroscopically the dimer–monomer dissociation equilibrium of human recombinant IFN-γ and have found that the monomers possess approximately 50% lower Trp quantum yield than the dimers [Boteva et al. Biochemistry 1996;35:14825]. In the present study we characterise the conformational properties of the two states — monomeric and dimeric, and analyse the effects of the salt composition of human blood plasma, physiological cations K⁺, Na⁺, Ca²⁺ and Mg²⁺ and mechanical stress on the dimer–monomer equilibrium. A medium with electrolyte composition of human blood plasma increases both the association and dissociation rate constants without shifting significantly the dimer–monomer equilibrium. The physiological cations shift the equilibrium towards dissociation of dimers into monomers by lowering the activation energy and the free energy of the process thus decreasing the stability of IFN-γ. Mechanical stress caused by stirring of the protein solution reduces irreversibly the Trp fluorescence by 75–80% and decreases significantly the -helical content and favours the aggregation.