Cloning, expression and bioinformatics analysis of 27 kDa cysteine protease gene of Spirometra erinacei plerocercoid / 中国人兽共患病学报

To clone and express 27 kDa cysteine protease (CP) gene of Spirometra erinacei plerocercoid,and analyze the biology characteristics,a total of RNA was extracted from the plerocercoids and reversely transcribed into cDNA.The 27kDaCP gene was amplified by PCR and cloned into pM-19T vector for sequencing.The accurate sequence was subcloned into the expression vector pET-28a (+).The recombinant plasmid was transformed into Transetta (DE3) and the expression protein induced by IPTG.The recombinant protein was purified by Ni2 + affinity chromatography,and analyzed by SDS-PAGE and Western blotting.The 27 kDa CP gene and its expression protein were predicted and analyzed by bioinformatics analysis tools such as NCBI and ExPASy.Results showed that the ORF length of 27 kDa CP gene sequence was 1 011 bp,and the removed signal peptide sequence was 954 bp with the submission number of ANA52569 in GenBank.The whole sequence of 27 kDa CP (Mr 35 669.9,pI 5.92) was 317 amino acids conferred from cDNA,which belongs to the Peptidase_C39_like superfamily.The pET-28a (+)-27kDa-CP was expressed under the induction of IPTG.Western blotting analysis showed that the purified protein reach expectancy,and had better response with positive serum of Spirometra erinacei plerocercoid infection.In conclusion,the 27 kDa CP gene of Spirometra erinacei plerocercoid is successfully cloned and expressed and knowing coded sequences and bioinformatic.

Protease gene shuffling and expression in Pichia pastoris

Four kinds of neutral and alkaline protease genes from Aspergillus oryzae and Bacillus subtilis were isolated and shuffled. The shuffled genes were selected, inserted into pGAPZαA plasmid and transformed into Escherichia coli. The gene which could express high-activity protease was selected by screening the sizes of transparent zones around the colonies on casein plates. After an ideal protease gene was selected, it was sequenced and then transformed into Pichia pastoris X33. The result showed that the base in 1022th position of shuffled protease gene was changed from thymine to cytosine, inferring that cysteine was changed to arginine in the mutant protease. After 48 h incubation for the transformed P. pastoris with the mutant or native protease genes, the mutant protease activity was 36.4% higher than the native protease (P<0.05). The optimal pH and temperature of the mutant protease were 6.5-8.0 and 30-70°C, respectively, which indicated better stability than the native protease (P<0.05).

High Frequency of Non-B Human Immunodeficiency Virus Type 1 (HIV-1) Subtype Specific Mutations at the Protease Gene among Treatment-naive HIV-1 Infected Individuals in Jos, Nigeria.

Aims: To determine the prevalence of non-B HIV-1 subtype specific mutations in the protease gene among antiretroviral drug-naive individuals in Jos, Nigeria. Study Design: This was a cross-sectional study in which randomly selected blood samples of HIV-1 positive anti-retroviral drug-naïve individuals were used for genotyping assay. Place and Duration of Study: The study was conducted at the adult HIV clinic of the AIDS Prevention Initiative in Nigeria (APIN) programme, Jos University Teaching Hospital (JUTH), Jos, Nigeria between October 2010 and April 2011. Methodology: Of the one hundred and five plasma samples, 100 samples were successfully reverse transcribed and amplified by nested PCR. The amplicons were directly sequenced on an automated ABI genetic analyzer using BigDye Terminator Cycle Sequencing Kit. Subtyping and phylogenetic analyses were performed using the REGA subtyping tool version 2.0 and MEGA 5.0 software. Both the Stanford HIV database algorithm and IAS-USA 2013 drug resistance update were used for interpretation of drug sensitivity. Results: The proportion of the non-B HIV-1 subtypes were as follows: CRF02_AG (48%), G (41%), CRF06_cpx (6%), A (5%). Q58E, a major drug resistance mutation to PI, occurred as a low prevalence mutation in subtype G. The most common mutations observed among the subtypes were I13V, K14R, K20I, M36I, R41K, H69K, V82I and L89M. Conclusion: A non-uniform distribution of non-B HIV-1 subtypes were observed in Jos, Nigeria, with CRF02_AG and G predominating among the antiretroviral drug-naive individuals. Among the different subtypes in circulation, there was a high prevalence of minor mutations and natural polymorphisms associated with the protease gene. Such mutations define the subtype diversity which may impact on virulence and drug ‘responses’, thus further studies are needed to evaluate the clinical implications of these mutations.

Expression and Purification of Herpes Simplex Virus Type 1 Protease

An attractive target for anti-herpes chemotherapy is the herpes simplex virus 1 (HSV-1) protease encoded by the UL26 gene. HSV-1 protease is essential for DNA packaging and virus maturation. To perform high throughput for potent inhibitors, the efficient production of larger amounts of highly purified enzyme and protease activity assay method must be established. In this report, expression in E. coli and purification of the protease gene of HSV-1 strain F was investigated. The protease gene was cloned pET28, and the nucleotide sequence of protease catalytic domain of HSV-1 compared strain F with other strains (KOS and CL101). In these results the F strain was different in base sequence. However, the amino acid sequence was identifical. The HSV-1 protease was purified with His-tagged affinity column. The analysis of HSV-1 protease activity Was performed by high performance liquid chromatography.

STUDY ON RESISTANCE GENE KNOCK OUT FROM INTEGRATED ALKALINE PROTEASE GENE ENGINEERING STRAIN / 微生物学通报

The knock out vector pHK was constructed with E coli vector pET 28a and shuttle vector pHY300PL, by using denatured DNA and homologous recombination technique, the kanamycin resistance gene ( Kan r) from integrated alkaline protease gene engineering strain BP071 was knocked out successfully, and the 11 positive clones were obtained The yield of the best positive clone BP0715 was stable as same as BP071 The methods provided the good experience for the industrial microbiology research, and it was foundation for studying on the safety of genetically modified organisms