Establishment of SeMNPV persistent infection in Spotoptera exigua cells / 病毒学报

Persistent baculovirus infection is observed frequently in insect populations. Persistent infection can be transformed to a replicative and infective state caused by stress factors and plays an important role in regulating the size of insect population and in epizoology of baculoviruses. The aim of this study is to establish a persistently baculovirus-infected cell system to explore the molecular mechanisms of baculoviral persistence. Spodoptera exigua nucleopolyhedrovirus (SeMNPV) was serially undiluted passaged in Se301 cells to reduce virulence. Upon infection of Se301 cells with the SeMNPV up to passage 8, a few cells survived even if most of cells died due to virus infection. The surviving cells were passaged and designated as P8-Se301 cell strain. P8-Se301 cells had a population doubling time of 58-65 hours and grew slower than Se301 cells. Light microscopy and electron microscopy observation showed symptom of baculovirus infection, such as virogenic stroma, viral particles and occlusion bodies, in some of P8-Se301 cells. End-point dilution assay and infectious center assay showed that 4.14% +/- 0.99% cells continually released infectious progeny virus which replicated slower than SeMNPV in Se301 cells. The result indicated that P8-Se301 cells show a typical character trait of baculovirus persistent infection.

Screening of Bacillus thuringiensis strains containing vip3A genes and analysis of gene conservation / 生物工程学报

Vip3A, a novel insecticidal protein, is secreted by Bacillus thuringiensis (Bt) during vegetative growth. Vip3A protein possesses insecticidal activity against a wild spectrum of lepidopteran insect larvae. Since the first cloning of vip3A gene from Bt, many other vip3A genes have been isolated. To investigate vip3A genes contribution to Bt and reflect the revolution relationships, the strains containing vip3A genes were screened and gene similarity was analyzed. 114 wild-type Bacillus thuringiensis (Bt) strains isolated from different regions and 41 standard Bt strains from the Institute of Pasteur were screened for the vip3A genes using PCR amplification. 39 strains including B. thuringiensis subsp. kurstaki (Btk) HD-1 were found to contain the vip3A genes. Because acrystallerous strain Cry- B derived from Btk HD-1 was proved not to contain vip3A gene, it suppose that the vip3A gene may be located at the plasmids. Vip3A proteins expressed in these strains were detected with polyclonal antibody by Western blot and 4 strains among them were shown not to express the Vip3A proteins. The vip3A genes amplified from wild-type Bacillus thuringiensis strains S101 and 611 with different levels of activity against lepidopteran insect larvae were cloned into pGEM-T Easy vector. Alignment of these 2 putative Vip3A proteins with 6 others (Vip3A (a), Vip3A(b), Vip3A-S, Vip3A-S184, Vip83 and Vip3V) in the GenBank data base and 2 reported Vip3A proteins (Vip14 and Vip15) showed that vip3A genes are highly conservative. The plasmids pOTP-S101 and pOTP-611 were constructed by in- serting 2 vip3A genes (vip3A-S101 and vip3A-611) into the expression vector pQE30 respectively and were transformed into E. coli M15. E. coli M15 cells harboring the pOTP plasmids were induced with 1 mmol/L IPTG to express 89 kDa protein. Experiments showed that the level of soluble proteins of Vip3A-S101 in E. coli M15[pOTP-S101] and Vip3A-611 in E. coli M15 [pOTP-611] were about 48% and 35% respectively. Bioassay showed that each of these Vip3A proteins had similar toxicity against neonate Spodoptera litura larvae, indicating that some amino acids change had little effect on the insecticidal activity of proteins. Although vip3A genes are conservative, the unknown insecticidal spectrum is still to be brought out. Vip3A genes can be used for the construction of the Bt engineered strains and transgenic plants. In addition, vip3A genes are excellent candidates for delay of the pest resistance due to the difference of the action model from that of Bt delta-endotoxins.

Bacillus thuringiensis helper protein P20 affects the formation of Cry1Ab / 生物工程学报

The Cry1Ab differs most significantly from the other related ICPs by its absence of a carboxyl terminus of 28 amino acids including four cysteines; consequently it is less stable. We report that the helper protein P20 plays a role in the expression and crystallization of Cry1Ab. Three Cry1Ab expression plasmids pT1B, pP1B, and pDP1B, were constructed based on the shuttle vector pHT3101. The vector pT1B does not contain the p20 gene, pP1B carries p20, and pDP1B contains p20 with cry1A(c) promoter. Transformants were obtained by electroporating the plasmids into Bacillus thuringiensis acrystalliferous mutant CryB. Western blot demonstrated that crylAb was expressed as a 130 kD protein in all the transformants, and some of the protein was partially degraded into a 60 kD peptide. Quantitative protein analysis indicated that the amount of the 130 kD protein varied in the transformants and was in the ratio of 1:1.4:1.5 for PT1B, pP1B and pDP1B respectively. For the 60 kD proteins, the ratio was 1:1.1:1.6. Microscopic examination revealed that the size of the typical pyramidal crystals in the three transformants was in the order of T1B < P1B < DP1B. Bioassay showed that T1B, P1B and DP1B were all toxic to the larvae of Helicoverpa armigera with similar LC50. This study suggested that P20 plays a role in the expression and crystallization of Cry1Ab.