To obtain recombinant human β2-microglobulin (rhβ2M) with properties of good solubility and high purity from E.coli, prokaryotic expression conditions were optimized and protein purification was performed in this study. After testing the effect of different IPTG concentrations, temperatures and induction times on the production of rhβ2M, the optimum expression conditions were determined, i.e. joining IPTG to final concentration being 0.8 mmol/L and inducing time 6 h and at temperature of 25℃. Under the optimum induction conditions, the ratio of soluble rhβ2M to soluble bacterial protein was 63.7%. After purified by Ni Sepharose 6 Fast Flow, the purity of rhβ2M achieved a greater value of 95%. Western blot analysis revealed that rhβ2M possessed the antigen property that specifically interacted with anti-β2M antibody.
In order to construct and express human cardiac troponin C-linker-troponin I(P) fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pColdⅠ-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni2+ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo FinecareTM cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37℃and 4 or more months at 25℃and 4℃. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.
Objective To investigate the clinical value of computed tomographic angiography (CTA) and three-dimensional reconstruction technique in repairing scalp avulsion wound with large skull exposure by the free latissimus dorsi flap transplantation. Methods Between October 2007 and June 2012, 9 female patients with serious scalp avulsion and large skull exposure were treated, aged 23-54 years (mean, 38 years). The injury causes included machine twist injury in 6 cases, traffic accident injury in 2 cases, and falling from height injury in 1 case. Before admission, 3 patients had scalp necrosis after scalp in situ replantation, and 6 patients underwent debridement and dressing. The time from injury to admission was 8 hours to 7 days (mean, 1 day). The avulsed scalp area ranged from 75% to 90% of the scalp area (mean, 81%); the exposed skull area ranged from 55% to 70% of the scalp area (mean, 63%). Two patients had unilateral auricle avulse. CTA was used to observe the superficial temporal artery and vein, facial artery, external jugular vein, dorsal thoracic artery and vein, and measure the blood vessel diameter before operation. According to the CTA results, the latissimus dorsal skin flaps were desinged to repair wounds in 7 cases, the latissimus dorsal muscle flaps combined with skin graft were used to repair wounds in 2 cases. According to preoperative design, operation was successfully completed in 7 cases; great saphenous vein was used as vascular graft in 2 cases having poor images of superficial temporal vessels. The size of latissimus dorsal skin flaps ranged from 20 cm × 14 cm to 25 cm × 20 cm; the donor site was repaired with skin graft. The size of latissimus dorsal muscle flaps were 23 cm × 16 cm and 16 cm × 10 cm; the donor site was directly sutured. Results The blood vessel diameter measured during operation was close to the value measured before operation. The operation time was 6-8 hours (mean, 6.5 hours). The latissimus dorsal muscle (skin) flap and skin graft survived, with primary healing of wound or incision at donor site. The patients were followed up 3 months-2 years (mean, 6 months). The flap had soft texture and skin had no ulceration. Conclusion The free latissimus dorsi flaps can repair scalp avulsion with large skull exposure. Preoperative CTA can get the vessel anatomical structure and diameter at donor and recipient sites, which will guide the operation program design and implementation so as to shorten the operation time and improve the accuracy rate of vascular anastomosis.