Cloning and expression of apolipophorin-III from the common cutworm,Spodoptera litura

We have cloned apolipophorin-III (apoLp-III) cDNA from adult fat body of Spodoptera litura. The sequence encodes a 188 amino acid polypeptide including a 22 amino acid leader peptide. The circular dichroism spectrum from the purified apoLp-III indicated a considerable content of α-helix. Sequence alignment showed that S. Litura apoLp-III has a relatively high degree of sequence identity with the apoLps-III of lepidopteran, Manduca sexta (72%), Galleria mellonella (67%), Bombyx mori (60%). These alignments with four lepidopteran apoLps-III showed highly identical residues and conservative replacements at a degree of 86%. Levels of mRNA from last instar larval fat body and adult fat body were compared through Northern blot analysis using ³²P-labeled 704 bp apoLp-III cDNA probe. A 850 bp mRNA was detected in both stages and mRNA level of day 1 adult fat body was much higher than that of last instar larval fat body. The tissue-distribution of apoLp-III mRNA in adult ovary and testis was also examined and we confirmed the presence of apoLp-III mRNA in ovary and testis although apoLp-III was expressed in these tissues at very low levels compared with the adult fat body. Arch. Insect Biochem. Physiol. 39:166–173, 1998. © 1998 Wiley-Liss, Inc.     

中国产家蚕抗菌肽A基因部分序列的测定

从大肠杆菌感染的家蚕蛹提取RNA,用RT-PCR方法扩增未知抗菌肽基因片段,经过克隆测序,获得了蚕抗菌肽A基因的部分片段164 bp,为制备蚕抗菌肽A基因探针,筛选基因文库打下了基础.     

Expression and characterization of inhA gene from Bacillus thuringiensis 8010

InhA, a zinc metalloprotease secreted by Bacillus thuringiensis, specifically hydrolyzes antibacterial peptides produced by insect hosts. In this study, the inhA gene was cloned from B. thuringiensis 8010 using a pair of degenerate primers and the deduced 796 amino acid sequence showed a high degree of similarity with other InhA proteins in the Bacillus cereus group. The deduced amino acid sequence contained the zinc-binding motif (HEXXH), which is characteristic of the zinc-metalloprotease family. Additionally, the inhA gene was expressed in Escherichia coli BL21 (DE3). The expressed InhA protein was shown to be toxic to the third larvae of Plutella xylostella, contrary to preliminary study concerning the effect of InhA on Bombyx mori. This study provided insights into the potential of InhA for the biological control of certain lepidopteran insects.     

Fusion expression of mutated cecropin CMIV in E. coli

A cDNA coding mutated cecropin CMIV fromBombyx mori was synthesized according to its amino acid sequence usingE. coli biased codons. The gene was cloned into the fusion expression vector pEZZ318 and was expressed inE. coli HB101. The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product. The anti-bacterial activities of recombinant cecropin CMIV were recovered after cleavage by chemical method.     

Expression pattern and tissue localization of the class B scavenger receptor BmSCRBQ4 in Bombyx mori

Class B scavenger receptors (SR‐Bs) are cell surface glycoproteins involved in various physiological processes in vivo, including the transport and metabolism of lipids, binding and phagocytosis of xenobiotics, and signaling. But little information is available about silkworm SR‐Bs; it is necessary to study these SR‐Bs for revealing their function. In this study, we cloned the full‐length coding sequence of BmSCRBQ4, a SR‐B gene from the silkworm Bombyx mori L. We found that the BmSCRBQ4 gene consists of nine exons and eight introns, with an open reading frame of 1371 bp encoding 456 amino acids. Gene expression studies determined that BmSCRBQ4 messenger RNA (mRNA) was expressed in unfertilized eggs, during embryonic development and throughout the majority of the larval period. Expression of mRNA was detected in the mid gut, middle silk gland, posterior silk gland, head, integumentum, fat body, testes and the ovaries of the larval B. mori Dazao strain, as well as in the silkworm cell lines BmN and BmE. Protein expression studies found BmSCRBQ4 protein was expressed only in the testes, fat body and middle silk gland of larvae, as well as in the silkworm cell lines BmN and BmE. The BmSCRBQ4 protein showed variability in banding patterns in different tissues and cells when analyzed by Western blotting. Immunohistochemical staining showed that the BmSCRBQ4 protein localizes to the constitutive membranes or cellular membranes of these tissues. These results indicated that BmSCRBQ4 gene may play some physiologically relevant roles at the cell surface in each tissue.     

Lipophorin and its Receptor in Lepidoptera

Lipophorin (Lp) has an approximate native molecular weight of 730 kDa for Bombyx mori and consists of ApoLp‐I and ApoLp‐II with molecular weights of 250 kDa and 90 kDa for B. mori and 230 kDa and 80 kDa for Hyphantria cunea and 230 kDa and 49 kDa for Lymantria dispar, respectively. Lipid in Lp was mostly composed of neutral lipid. Lp of B. mori maintains constant level during larval and pupal stages but greatly increases during adult stage in both male and female. Lp of H. cunea appeared in great amounts in protein yolk bodies of ovary when vitellogenesis is actively taking place and was present in testicular fluid but not in the peritoneal sheath and cysts of testis. ApoLp‐III of B. mori has a molecular weight of 17 kDa and similar amino acid composition as those of other species Lp. H. cunea apoLp‐III has a molecular weight of 18 kDa and was present in all stages and in the protein body of ovary and in the cyst of testis. ApoLp‐III is synthesized in larval and adult fat body. cDNA sequence of Spodoptera litura apoLp‐III encodes a 188 amino acid polypeptide including a 22 amino acid leader peptide. Galleria mellonella Lp receptor has an approximate molecular weight of 97 kDa and 110 kDa under non‐reducing and reducing conditions, respectively and bound HDLp specifically. Lp receptor cDNA of G. mellonella showed th pattern of the VLDL receptor belonging to the LDL receptor family. The variant Lp receptors were expressed in the fat body of G. mellonella; one is a Lp receptor which lacks 84 bp of O linked sugar domain and the other is a full length form of the Lp receptor. The Lp receptor from the fat body of G. mellonella was differently expressed depending on the tissue and the developmental stages with specific abundance in prepupal stage.     

Distribution of Methanotrophs in the Phyllosphere

A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by genomic Southern hybridization with a probe of a DNA fragment synthesized by PCR. This was done using primers constructed based on the N-terminal amino acid sequence of a polygalacturonase, protopectinase-AS, produced by the strain and the consensus internal amino acid sequence of fungal polygalacturonases. The cloned polygalacturonase gene, containing an ORF, encodes 362 amino acids, including a 52-bp intron. It contains the consensus nucleotide sequence of PacC binding sites, and its expression was appeared to be regulated by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharomyces cerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase produced was the product of the cloned gene.     

Identification and characterization of Japanese encephalitis virus envelope protein gene from swine

Aims: Identification and characterization of Japanese encephalitis virus (JEV) envelope protein gene from swine. Methods and Results: Genomic RNA was separated from JEV isolated strain Henan‐09‐03, and used as templates for cDNA synthesis of E gene. The cDNA of E gene was amplified by RT‐PCR and cloned into the pMD19‐T‐Vector and confirmed by sequencing. The cloned gene was then subcloned into the pET‐32a and was introduced into Escherichia coli BL21 (DE3) for expression. The E protein was purified by Ni chelating column‐based affinity chromatography. The molecular weight of expressed protein was about 50 kDa. Compared with the published sequence of SA14 ( AF495589 ), the homology of the nucleotide sequence was 98% and the seven mutations resulting in amino acid substitutions at Leu 36 Ser, Leu107 Val, Ala167 Thr, Asn 230 Ser, Leu 340 Pro, Asn 430 Ile, Phe 448 Leu. Phylogenetic analysis of the E sequence of isolated strain classified it within genotype III of the JEV. The result of Western blotting indicated that the antigenicity of the protein was specific. Conclusions: The stable expression of the protein and the analysis of its antigenic specificity provide the foundation for developing the ELISA early stage diagnosis kit. Significance and Impact of the Study: As coating antigen, the recombinant E protein served a good source in the indirect ELISA method for the detection of JEV antibody.     

Cloning of exoinulinase gene from Penicillium janthinellum strain B01 and its high-level expression in Pichia pastoris

Aims: The aim of this study is to improve exoinulinase production by expression of a cloned exoinulinase gene inuA1 (GenBank accession no. JF961344 ) from Penicillium janthinellum strain B01 in Pichia pastoris. Methods and Results: A full‐length cDNA of exoinulinase gene (inuA1) was cloned from P. janthinellum strain B01 using RACE PCR. An open reading frame (ORF) of 2115 bp is interrupted by a single intron of 67 bp. The fragment encodes a signal peptide with 20 amino acids and a mature protein with 684 amino acids. The inuA1 was subcloned to the pPICZαC expression vector and succesfully over‐expressed in Pichia pastoris X‐33. The highest activity of exoinlinase reached 272·8 U ml^?1 in the fermentation liquid. It was c. 11‐fold of that produced by wild‐strain B01. A large amount of fructose was identified after the hydrolysis of inulin with the crude recombinant exoinulinase. The recombinant exoinulinase was purified and characterized. The molecular weight of the purified recombinant exoinulianse was 100 kDa. The mass spectrometry result indicated that the purified protein was indeed recombinant exoinulinase. The optimal pH and temperature of the purified recombinant exoinulianse were 4·5 and 50°C, respectively. Conclusions: An exoinulinase gene of P. janthinellum strain B01 was cloned, sequenced and over‐expressed successfully in P. pastoris. Significance and Impact of the Study: Only a few genes have been cloned from P. janthinellum because its molecular biology is poorly understood. In this study, we cloned and over‐expressed inuA1 gene of P. janthinellum in P. pastoris. This recombinant exoinulinase can be used to hydrolyse inulin to produce fructose and facilitate the biofuel production from inulin resources.     

Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus

The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. ). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M(r) of 11 kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11 kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with K(i) value of 0.1 and 0.6 nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294 bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis.     

Cloning of the Glyceraldehyde 3‐phosphate Dehydrogenase Gene of Flavescence dorée Phytoplasma and Development of Serological and Molecular Tools for Studying its Expression

The glyceraldehyde 3‐phosphate dehydrogenase (gapA) gene codes for a protein involved in the glycolytic pathway and is commonly used in Real‐Time RT‐PCR quantification studies as housekeeping gene. In this work we cloned and sequenced the full‐length gapA gene from Flavescence dorée phytoplasma (FDp). A ～35 kDa recombinant GapA protein was over‐expressed in Escherichia coli, purified and used as antigen to raise anti‐GapA rabbit polyclonal antibodies. The antiserum detected the GapA protein by western blot analysis of total protein extracts of FDp‐infected experimental host (Catharanthus roseus) and grapevine plants collected in the field. We also developed an FDp‐specific gapA Taqman Real‐Time RT‐PCR assay suitable for quantification overtime of gapA mRNA in infected plants.     

ANALYSIS OF HELICOVERPA ARMIGERA SINGLE NUCLEOPOLYHEDROVIRUS IMMEDIATE EARLY 1 (IE‐1) GENE*

Abstract A 6.12 kb Xbal‐H fragment of the Helicoverpa armigem single nucleopolyhedrovirus (HaSNPV) gemone was cloned and the complete sequence of this fragment was sequenced by random sequencing method. Sequence comparison and analysis revealed an ORF13 which was homologous to ie‐1 of Auiographa California nucleopolyhedrovirus (AcMNPV). The homologous encoding gene is ie‐1. The total length of the encoding region of HaSNPV gene was 1986 bp and was predicted to encode 661 amino acid protein(IE‐1) with molecular weight of 76.5 kD. The alingment of putative HaSNPV IE‐1 amino acid sequence with those of other 9 reported baculoviruses IE‐Is showed that the HaSNPV IE‐1 was most closely related to Helicoverpa zea nucleopolyhedrovirus (HzNPV) IE‐1, with 97% amino acid identidy. But it showed a low degree of sequence similarity to those of AcMNPV, Bombyx mori nucleopolyhedrovirus (BmNPV), Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV), Lymantria dispar nucleopolyhedrovirus (LdMNPV), Orgyia pseudotsugata nucleopolyhedrovirus (OpMNPV), Spodoptera exigua nucleopolyhedrovirus (SeMNPV), Plutella xylostella granulovirus(PxGV) and Xestia c‐nigrum granulovirus (XcGV), with 23%, 23%, 23%, 25%, 23%, 14%, 27% and 7% amino acid identity, respectively. A phylogenetic tree of ten baculoviruses IE‐1 was also given.     

Cloning and expression of chondroitinase AC from Bacteroides stercoris HJ-15

Enzymes that degrade glycosaminoglycans (GAGs) can help reveal the biological roles, structure, and mechanisms of GAGs. We cloned chondroitinase AC, which can degrade chondroitin sulfates A and C, from the genomic library of Bacteroides stercoris HJ-15 isolated from human intestine. The probe (1.4 kb) for the chondroitinase AC gene was prepared from the PCR product of the primers produced using two internal amino acid sequences of chondroitinase AC purified from B. stercoris HJ-15. Using this probe, a chondroitinase AC-positive, 4 kb DNA fragment was selected from pKF3 vector gene libraries containing 2.5–4.5 kb DNA fragments digested with HindIII. The amino acid sequence of the cloned chondroitinase AC showed 41% homology to that of Flavobacterium heparinum. The cloned chondroitinase AC gene was expressed under the T7 promoter of the expression vector, pET-26b(+), in Escherichia coli BL21(DE3) and purified using His bind column chromatography. The expressed chondroitinase AC potently degraded chondroitin sulfates A and C.