[Construction of a multi-copy Pichia expression vector and expression of human brain-derived neurotrophic factor in Pichia pastoris].

A pPICZ alpha1 vector was reconstructed with pPICZ alpha vector and PCR product of His4-Kan sequence from pPIC9K vector. The sequence encoding human brain-derived neurotrophic factor (hBDNF) was inserted into pPICZ alpha1. The expression vectors carrying 1, 2, 3, 6 copies of expression cassettes were constructed and transformed into Pichia pastoris GS115 respectively. The transformants were screened with G418/Zeocin and induced by 0.5% methanol, and the results of SDS-PAGE confirmed that the expression protein was about 14 kD and the percentage of the protein was about 20% of the total secreted protein. ELISA and Western blot analysis demonstrated that the protein could be specifically combined with chicken antibodies to human-BDNF.

[The construction of the hammerhead ribozyme genes targeting against apple scar skid viroid and its activity detection in vitro].

The genes of short armed hammerhead ribozyme targeting against two sites on positive strand (194-196) and negative strand (89-91) of ASSVd were designed, synthesized and cloned according to the action manner of hammerhead ribozyme. The full lengths of the genes are 42 bp (RzASSVd(+)) and 40 bp (RzASSVd(-)). After transcription in vitro, the ASSVd positive and negative RNA labeled with 32P were mixed with the ribozyme transcript and incubated 3-4 h at 50 degrees C or 37 degrees C. The results were assayed on 8% PAGE (containing 8 mol/L urea) and autoradiogrammed. As predicted, the transcript of the active RzASSVd(-) could cleave the ASSVd negative strand RNA with a high activity but had no cleavage effect on the ASSVd positive strand. The transcript of the RzASSVd(+) gene could cleave the ASSVd positive strand but its cleavage activity was very low. As the same, it cannot cleave the negative strand either. On the base of the result, we construct dimmer ribozyme gene pGEMRzASSVd(+/-) containing both RzASSVd(+) and RzASSVd(-).

[Design and preparation of the multimeric self-cleavable hammerhead ribozyme targeting apple scar skid viroid and its activity detection in vitro].

A self-cleaving hammerhead ribozyme gene containing a 14nt target sequence of ASSVd at the 3' end of hammerhead ribozyme was synthesized, amplified and cloned at the Xho I-Hind III site of pGEM7Zf(+). The ends produced by Xho I or Sal I can link together, thus the recognition sites of both enzymes vanish and can't be cut by either one. We used this property to get the recombinant plasmid bearing 2, 4, 6, 8, 10 and 12 copies of self-cleavable ribozyme respectively after successively sub-cloning five times. Linearized recombinat plasmid model catalyzed by T7 RNA polymerase was transcribed in vitro. The multimeric ribozyme molecules efficiently self-cleaved via cis-acting to release many ribozyme molecules It indicates that the concentration of ribozyme transcripts has been enhanced during transcription. Trans-cleavage reaction was carried out by incubating monomeric and multimeric ribozymes with same mol concentration and 32P labeled target ASSVd. Both ribozymes and target transcripts were mixed in 1:1 ratio. Autoradiograms showed the transcripts of multimeric ribozyme were substantially more effective against the ASSVd target RNA than the monomeric ribozymes. We confer that the multimeric self-clevable ribozyme is likely to provide more valuable application in vivo.