Amino acid sequences and disulfide bridges of serine proteinase inhibitors from bitter gourd (Momordica charantia LINN.) seeds

Three serine proteinase inhibitors, MCTI-I, MCTI-II, and MCEI-I, were isolated from bitter gourd (Momordica charantia LINN.) seeds. MCTI-I and MCTI-II were inhibitors for trypsin and MCEI-I was an elastase inhibitor. Their amino acid sequences and the positions of disulfide bridges of MCTI-II were determined to be as follows. (sequence; see text)     

Effect of P(2)' site tryptophan and P(20)' site deletion of Momordica charantia trypsin inhibitor II on inhibition of proteinases

Momordica charantia trypsin inhibitor II (MCTI-II) inhibits the amidolytic activity of factor Xa with a K(i) value 10-100-fold smaller than those of other squash family inhibitors. It also inhibits factor X activation mediated by factor VIIa-tissue factor complex or factor IXa. Comparison of other squash family inhibitors reveal Trp at position 7 (P(2)') and a deletion at position 25 (P(20)') are characteristics of MCTI-II. In order to elucidate the effect of these positions on the inhibitory activity, we chemically synthesized three inhibitors: S-MCTI-II whose amino acid sequence is identical to natural MCTI-II, S-MCTI-II(7L) whose P(2)'(Trp) is substituted with Leu, and S-MCTI-II(25N) whose P(20)'(deletion) is filled with Asn. The dissociation constants of the complexes of human factor Xa with S-MCTI-II, S-MCTI-II(7L), and S-MCTI-II(25N) were 1.3x10(-6) M, 2.8x10(-5) M, and 7.3x10(-6) M, respectively. They inhibited factor X activation mediated by factor VIIa with the same degree. As in the case of natural MCTI-II, S-MCTI-II suppressed factor X activation mediated by factor IXa, while S-MCTI-II(7L) and S-MCTI-II(25N) did not. Both the Trp at the P(2)' position and deletion at the P(20)' position are thus likely required for the inhibition of factor Xa, trypsin, and factor IXa, while these two positions do not affect factor X activation initiated by the factor VIIa-tissue factor complex.     

Momordica charantia trypsin inhibitor II inhibits growth and development of Helicoverpa armigera

Abstract  Bitter gourd ( Momordica charantia L.) seeds contain several squash-type serine proteinase inhibitors (PIs), which inhibit the digestive proteinases of the polyphagous insect pest Helicoverpa armigera . In the present work isolation of a DNA sequence encoding the mature peptide of a trypsin inhibitor McTI-II, its cloning and expression as a recombinant protein using Pichia pastoris have been reported. Recombinant McTI-II inhibited bovine trypsin at 1: 1 molar ratio, as expected, but did not inhibit chymotrypsin or elastase. McTI-II also strongly inhibited trypsin-like proteinases (81% inhibition) as well as the total proteolytic activity of digestive proteinases (70% inhibition) from the midgut of H. armigera larvae. The insect larvae fed with McTI-II-incorporated artificial diet suffered over 70% reduction in the average larval weight after 12 days of feeding. Moreover, ingestion of McTI-II resulted in 23% mortality in the larval population. The strong antimetabolic activity of McTI-II toward H. armigera indicates its probable use in developing insect tolerance in susceptible plants.     

人白细胞介素-11基因在家蚕培养细胞和虫体内的表达

将缺少编码信号肽序列的人白细胞介素－11（hIL-11)546核苷酸cDNA,重组于质粒pBacPAK8构建重组转移载体pBacIL-11,与经线性化修饰的家蚕核型多角体病毒（BmBacPAK)DNA共转染家蚕培养细胞株BmN,获得了插入hIL-11基因的重组病毒。Southern杂交表明重组病毒基因组中含有hIL-11基因片段,RNA斑点杂交表明hIL-11基因得到了转录。重组病毒感BmN细胞株、家蚕幼虫和蛹,在细胞培养上清、细胞抽提物、幼虫和蛹的体液样品中,SDS－PAGE电泳分析都能检测得到表达产物的特异性条带;采用IL－11依赖细胞株B9－11和MTT法测定表达产物的生物活性,表明rIL-11基因分别在培养细胞和蚕体内得到了高效表达。     

植酸酶基因在家蚕—杆状病毒表达系统中的表达及其酶学特性

将从黑曲霉菌株Aspergillusniger 96 3克隆并经改造后的植酸酶基因在昆虫 -杆状病毒表达系统中表达 ,SDS PAGE电泳检测蚕体和蛹的表达量分别达到 1.4 3g L血淋巴液和 1.90g L血淋巴液。酶活性测定结果表明 ,在蚕体和蛹的表达活性分别为 4 .6 7× 10 8u L血淋巴液和 5 .99× 10 8u L血淋巴液。该酶活性的最适温度范围为 5 0～6 0℃ ,最适pH值为 5 .5～ 5 .0和 2 .5。研究表明杆状病毒系统表达的植酸酶具有耐酸性和抗高温的特性 ,可以用于生产饲用植酸酶。     

Biosynthesis of recombinant human hepatitis B M-HBsAg in silkworm larvae

The middle surface antigen (M-HBsAg) of human hepatitis B virus is virus envelope protein. It's used as a basis for development of vaccine and test-system for detecting of hepatitis B virus. The cDNA of M-HBsAg was inserted into transfer vector pBK273 under the polyhedron promoter with obtaining of recombinant plasmid DNA pBHep-2. As a result of cotransfection pBHep-2 with wild type BmNPV the recombinant baculovirus rBmNPVHep which included the cDNA of M-HBsAg under the polyhedron promoter was obtained. Infection of silkworm larvae Bombyx mori with recombinant virus resulted in expression of foreign gene and accumulation of middle surface antigen of human hepatitis B virus mostly (>90%) in fat bodies of silkworm larvae.     

Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor

To date, many recombinant proteins have been expressed in Bombyx mori cells or silkworm larvae, apart from in pupae. Silkworm pupae may be more suitable for the expression of heterologous proteins as a bioreactor. If maintained at an appropriate temperature, silkworm pupae could be inoculated with recombinant baculovirus for the expression of a protein of interest. In this study, human granulocyte-macrophage colony-stimulating factor was successfully expressed in silkworm pupae using B. mori nucleopolyhedrovirus, purified and characterized with respect to its physico-chemical properties. The target protein expressed had an apparent molecular mass of 29 kDa and an isoelectric point of 5.1. The protein was purified using three chromatographic steps with a final recovery of 10.3%. Finally, approximately 3.5mg of the protein was obtained with a biological activity of up to 8.4 x 10(6) cfu mg(-1). The results of this study suggest that silkworm pupae represent a convenient and low-cost bioreactor for the expression of heterologous proteins.     

A new technique for producing recombinant baculovirus directly in silkworm larvae

A new technique for the direct production of recombinant baculovirus in the silkworm larvae is described. To assess the utility of this method, a combination of Bombyx mori nucleopolyhedroviral genome, transfer vector and Lipofectin was co-injected directly into newly ecdysed fifth instar, silkworm larvae. The recombinant virus was obtained from the hemolymph of injected larvae and the hemolymph then re-injected into the larvae as an inoculum. This resulted in a high-level production of foreign protein in the silkworm larvae. This technique produces easy and rapid recombinant protein production in silkworms.     

人GDNF在甲醇酵母及家蚕幼虫中的表达

将人胶质细胞源性神经营养因子(GDNF)基因克隆入酵母分泌型表达载体pPIC9K中,酶切线性化后电穿孔导入酵母细胞进行整合,经G418筛选得到多拷贝转化子,甲醇诱导表达。将人GDNF基因克隆入昆虫病毒转移载体pBacPAK8中,与线性化Bm-BacPAK6修饰病毒基因组DNA共转染家蚕细胞,经体内重组,筛选到重组病毒。用重组病毒感染家蚕幼虫,5d后收集血淋巴。SDS-PAGE和蛋白质印迹杂交结果证实了酵母培养上清液及家蚕幼虫血淋巴中含有GDNF蛋白。活性研究表明,甲醇酵母及家蚕幼虫表达的GDNF蛋白能促进多巴胺能神经元的存活和突起生长。     

Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm

Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.     

HaSNPV几丁质酶基因在大肠杆菌和昆虫细胞中的表达及其产物对Bt杀虫剂的增效作用

为了探索杆状病毒几丁质酶对微生物杀虫剂的增效作用及其利用途径 ,分别在大肠杆菌和昆虫细胞中表达棉铃虫单粒包埋型核型多角体病毒 (HaSNPV)几丁质酶 .用PCR方法扩增出不含N端信号肽编码序列的几丁质酶基因片段 ,并分别克隆至原核表达载体pET2 8a和重组到杆状病毒BactoBac表达系统 ,在大肠杆菌 (E .coli)BL2 1和粉纹夜蛾 (Trichoplusiani)细胞系Tn 5B1 4中分别进行了表达 .在大肠杆菌中表达量约占细菌总蛋白 15 % ,在昆虫细胞中表达量约占细胞总蛋白10 % .将含有几丁质酶的大肠杆菌和昆虫细胞表达产物添加到苏云金杆菌 (Bt)菌液中一起喂食 2龄家蚕 .结果显示 ,HaSNPV几丁质酶基因的 2种表达产物和Bt杀虫剂的混合物使处理的家蚕的致死时间较对照处理均明显缩短 .昆虫细胞和大肠杆菌表达产物与Bt混合物处理的LT50 分别从 93 5h和 95 1h缩短到 5 6 2h及 6 7 2h ,并且供试家蚕的生长速度明显缓慢 .研究结果表明 ,重组的HaSNPV几丁质酶有望作为Bt杀虫剂的增效剂     

Expression of UreB and HspA of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in silkworm pupae and identification of its immunogenicity

For mass production of urease B subunit (UreB) and heat shock protein A subunit (HspA) of Helicobacter pylori with Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) and to determine whether they could be used as an oral vaccine against H. pylori, besides, to determine the time course of expressed recombinant protein and the optimum acquisition time directly through green fluorescence, HspA and enhanced green fluorescence protein (EGFP) genes were cloned into vector pFastBacDual to form donor vector pFastBacDual-(EGFP) (HspA), UreB gene was cloned into vector pFastBacDual to form donor vector pFastBacDual-UreB,then they were transformed into E. coli BmDH10Bac to obtain the recombinant Bacmid-(EGFP) (HspA) and Bacmid-UreB respectively. They were used to transfect BmN cells and generated the recombinant baculovirus BmNPV-(EGFP) (HspA) and BmNPV-UreB. Using these recombinant baculovirus BmNPV-(EGFP) (HspA) and BmNPV-UreB inoculated the silkworm pupae, a recombinant HspA and UreB protein were expressed in silkworm pupae, which were around 13 and 62 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. After oral immunization of mice, serum specific IgG antibodies against HspA and UreB in vaccine group were much higher than that in mock and native silkworm powder control groups. The results indicated that the expressed recombinant HspA and UreB in silkworm pupae would possess good immunogenicity. In addition, when EGFP and HspA proteins were expressed, a direct correlation between the increase in intensity of fluorescence and HspA concentration.     

Protein engineering of novel proteinase inhibitors and their effects on the growth of Spodoptera exigua larvae.

Novel types of proteinase inhibitors with multi-inhibitory activities were generated by replacement of phytocystatin domains in sunflower multi-cystatin (SMC) by the serine proteinase inhibitor BGIT from bitter gourd seeds. Two chimeric inhibitors SMC-T3 and SMC-T23, in which the third domain in SMC and the second and third domains in SMC were replaced by BGIT, acquired trypsin inhibitory activity (Ki: 1.46 x 10(-7) M and 1.75 x 10(-7) M), retaining inhibitory activity toward papain (Ki: 4.5 x 10(-8) M and 1.52 x 10(-7) M), respectively. We compared the chimeric inhibitors and the recombinant SMC (r-SMC) in relation to their effects on the growth of larval Spodoptera exigua. When the second instar larvae were reared on a diet containing rSMC, SMC-T3, or SMC-T23 for ten days, a significant reduction in weight gain was observed. Mean weights for rSMC, SMC-T3, and SMC-T23 were 43 mg, 32 mg, and 43 mg, respectively, as compared with that (60 mg) for the absence of the inhibitor. In contrast, BGIT had little effect on the growth of the S. exigua larvae. This result indicated that the chimeric inhibitor SMC-T3 with two phytocystatin domains and one serine proteinase inhibitor domain is an efficient inhibitor of proteinases in the S. exigua larvae. Therefore, this novel type of proteinase inhibitor with multi-inhibitory activities may represent a promising protein for successful application to a transgenic plant with insect resistance.     

Improvement of recombinant baculovirus infection efficiency to express manganese superoxide dismutase in silkworm larvae through dual promoters of Pph and Pp10

The silkworm, Bombyx mori, has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). There are several problems which will probably be the bottleneck for practical and industrial utilization of silkworm bioreactor. Traditionally, the recombinant virus should infect the larvae through individual dorsal injection by a syringe. This is a time- and labor-consuming procedure. This drawback has become a bottleneck for practical and industrial utilization of baculovirus expression system in the silkworm bioreactor. In this paper, we constructed a dual expression baculovirus to express the renovated polyhedron and target manganese superoxide dismutase (SOD) gene under P10 and polyhedron promoters, respectively, through oral infection. The results showed that the direct injection of recombinant rBacmid/BmNPV/SOD DNA with cellfectin reagent infected the silkworm larvae partially. When next batches of larvae were fed orally with hemolymph, which was collected from first batch of injected and infected larvae, the obvious symptom of infection was found and high target SOD was expressed. These results imply it is feasible to express target genes through combination of recombinant bacmid DNA injection and oral feeding by a dual expression bacmid baculovirus.     

Isolation of a trypsin inhibitor with deletion of N-terminal pentapeptide from the seeds of Momordica cochinchinensis, the Chinese drug mubiezhi.

A trypsin inhibitor, MCCTI-1, with a molecular weight of 3479 Da as determined by mass spectrometry, was isolated from Momordica cochinchinensis seeds with a procedure involving extraction with 5% acetic acid, ammonium sulfate precipitation, ion exchange chromatography on CM-Sepharose and reverse-phase high performance liquid chromatography. The sequence of its first 13 N-terminal amino acid residues was ILKKCRRDSDCPG which was about 85% identical with the sequence of trypsin inhibitor MCTI-1 from Momordica charantia Linn. When compared with the sequences of most other squash family trypsin inhibitors, the sequence of MCCTI-1 was characterized by the deletion of a pentapeptide from the N-terminus. Trypsin inhibitors also existed in seeds of some hitherto uninvestigated Cucurbitaceae species.     

Production of recombinant Bombyx mori nucleopolyhedrovirus in silkworm by intrahaemocoelic injection with invasive diaminopimelate auxotrophic Escherichia coli containing BmNPV-Bacmid

The present study elaborates a cost-effective and transfectant-free method for generating recombinant Bombyx mori (silkworm) nucleopolyhedrovirus in silkworm larvae and pupae by injecting invasive Escherichia coli carrying BmBacmid [BmNPV (B. mori nucleopolyhedrovirus)-Bacmid] into larval haemocoel. Up to 109 PFU (plaque-forming units)/ml of infective recombinant baculovirus was generated in the silkworm by intrahaemocoelic injection with 106 DAP (diaminopimelic acid) auxotrophic and BmBacmid containing E. coli cells expressing both invasin and listeriolysin. Thus 1?ml of overnight culture of E. coli is sufficient to inject more than 2000 larvae, while DAP costing up to $1 is enough to inject about 4000 larvae. Recombinant proteins can be controlled to be expressed mainly in pupae by adjusting the injection dose, too. In this new method, many original manipulations have been eliminated, including BmBacmid preparation and the subsequent complex transfection procedures. Hence it is a time- and cost-saving means for large-scale injection of B. mori for recombinant baculovirus production in comparison with the traditional transfection methods, which may play an important role in the industrial development of the BmNPV-silkworm bioreactor.     

Baculoviral infection reduces the expression of four allergen proteins of silkworm pupa

Silkworm (Bombyx mori) larvae are widely used to express exogenous proteins. Moreover, some silkworm pupal proteins can be used as drug‐loading materials for selfexpressed oral tolerance drugs. However, several proteins expressed in silkworm pupae cause severe allergic reactions in humans and animals. Interestingly, some baculovirus vectors have been shown to alter the host gene and its expression in insect cells, but this has not been confirmed in silkworm. Here, we analyzed the effects of infection with an empty B. mori baculovirus (BmNPV) vector on silkworm pupal protein expression. Using a proteomics approach, the allergens thiol peroxiredoxin (Jafrac1), 27‐kDa glycoprotein (p27k), arginine kinase, and paramyosin as well as 32 additional differentially expressed proteins were identified. Downregulation of the messenger RNA expression of the four known allergens was observed after BmNPV infection; subsequent changes in protein expression were confirmed by the western blot analysis using polyclonal antibodies prepared with recombinant proteins of the four allergens. Collectively, these data indicate that the four known allergens of silkworm pupae can be reduced by infection ith an empty BmNPV vector to increase the safety of silkworm pupa‐based exogenous protein expression and drug delivery of oral pharmaceuticals. In addition, the four recombinant allergen proteins may contribute to the diagnosis of allergic diseases of silkworm pupa.     

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。     

家蚕溶茧酶基因的真核表达及其产物的生物活性检测

为了研究家蚕 Bombyx mori 溶茧酶基因 （cocoonae）真核表达及其产物的生物活性,将溶茧酶基因（GenBank 登录号 EF428980）克隆至杆状病毒转移载体 pFastBac^TM 1 中获得 pFast-cocoonase,将其转化 DH10Bac 感受态细胞后,PCR 方法检测证实所分离的重组病毒 DNA 中含有目的片段溶茧酶基因。用脂质体法 转染家蚕 BmN 细胞,获得重组病毒。SDS-PAGE 分析显示,感染重组杆状病毒 Bac-cocoonase 的细胞表达产物在约为 27.6 kD 处出现特异性条带,这与预测的蛋白大小相符。用该表达产物与茧丝反应后,电镜下观察茧丝的形态,结果表明表达产物对茧丝的丝胶层有一定的水解作用。     

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.