The HIS tag purification system is used to improve the production of recombinant protein and to simplify production technique.HSP65-6P277 was a kind of recombinant protein which prevented the type 1 diabetes.Here,a study with a purpose of high level expression and convenient procedure of this vaccine in Escherichia coli was presented.The 6 × His-tag was artificially added at HSP65-6P277 N-terminal using PCR(polymerase chain reaction) amplification technique.Then this gene cassette was cloned into pET28a and the HIS affinity tagged HSP65-6P277 fusion protein expression plasmid pET28a-HIS-HSP65-6P277 was successfully constructed.By lactose induction,the fusion protein HIS-HSP65-6P277 was expressed as soluble form in the E.coli BL21(DE3).The recombinant HIS-HSP65-6P277 was purified only by HIS Bind Resin affinity chromatography.The electrophoretic behavior in SDS-PAGE and the result shown in the Weston blotting illustrated that the purified HIS-HSP65-6P277 was a HIS tag fusion protein with molecular weight of 70 k.As a result,the time period of the whole production technique was reduced from 15 ～ 20 days to 2 ～ 3 days.And the production was increased from 12 mg to 56 mg per liter culture.In conclusion,the HIS tag purification system provides a novel approach to simplifying the production technique and amplifying production of a vaccine against type 1 diabetes.

OBJECTIVE To evaluate the therapeutic effect of a novel recombinant human parathyroid hormone related-peptide on ovariectomized rats. METHODS Sixty-four of 88 3-month-old female Sprague-Dawley rats were ovariectomized and 16 received sham operation.Three months later,8 rats from each group received bone material density (BMD) test. Then the remaining 56 ovariectomized rats were randomly assigned to 7 groups:control group (NS,0.5 mL normal saline),8L-hPTH groups (20,10,5 μg·kg-1),hPTH(1-34) groups (20,10,5 μg·kg-1). Another 8 rats without any operation and sham-operated rats (sham) were treated in the same way as those of group NS. All rats were subcutaneously injected in consecutive days for 3 months. The femoral and lumbar BMD were measured and the femurs were observed by scanning electron microscope after 3-month-therapy. RESULTS Three months later,the femoral and lumbar BMD of ovariectomized rats were significantly decreased,compared with those of sham-operated rats (P0.01). Furthermore,the femoral and lumbar BMD and trabecular widths of 8L-hPTH therapeutic groups were all increased obviously compared with those of group NS (P0.05 and P0.01). CONCLUSION 8L-hPTH is effective in stimulating bone formation and improving skeletal microarchitecture. 8L-hPTH is potentially a effective therapeutical agent on osteoporosis.

A new fusion expression vector, pED-P8-Hi-lys was designed and constructed. It includes four parts, a 20 peptide sequence of hirudin that can maintain anticoagulant activity, the C-terminus of asparaginase as a fusion partner, basic octopeptide (KRKRKKSR) that makes the fusion partner easy to remove, and the unique acid-labile aspartyl-prolyl bond. It was transformed into E. coli BL-21 and the fusion protein (AnsB-C-P8-Hi-lys) was expressed effectively as inclusion bodies after inducing by lactose. The objective peptide Hi-lys was purified by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, and DEAE-cellulose 52 column chromatography. The antithrombin activity of the purified Hi-lys peptide was about 50 ATU/mg by thrombin activity assays.