Avicenna Journal of Medical Biotechnology
Volume:5 Issue: 3, Jul-Sep 2013

Over the past several years, mammals have been successfully cloned by either the splitting of an early stage embryo or nuclear transfer of adult somatic cells (NT) into oocytes. Although it has been 15 years since the generation of the first cloned mammals from somatic cells by NT, the success rate for producing live offspring by this technique is low regardless of the cell type and animal species used. However, these techniques have the potential to be important tools for future research in basic biology. In the present study, we described our experiences in producing successfully cloned mouse using NT method and piezo-actuated micromanipulator. B6D2F1 mice, 8-12 weeks old, were superovulated with injections of 5 IU of pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotropin administered 48 hr apart. Enucleation and donor nuclei cumulus cell injection were performed with a piezo-actuated micromanipulator after which activation and trichostatin A treatment were used for reconstructed oocytes. Two-cell stage cloned embryos that developed in the mWM medium were transferred into the oviducts of pseudopregnant NMRI mice. Of 367 oocytes collected, 131 (69%) developed into 2-cell stage embryos. Of these, 5 (1%) live pups were successfully delivered. We used NMRI foster mother to raise the pups by lactation. One adult cloned mouse was mated, after which she delivered and raised normal offspring. For mouse cloning, the present study also successfully tested the capability of somatic cell nuclear transfer SCNT using a piezo unit.

Recombinant monoclonal antibodies have been marketed in last three decades as the major therapeutic proteins against different cancers. However choosing a proper medium and supplements to reach the high expression is a challenging step. Despite of commercial serum free and chemically defined media, there are still numerous researches seeking the optimum media to gain higher expression titer. Selecting the best basal media followed by proper supplementation to increase the cell density and expression titer needs proper and accurate investigation. In this study, we have determined the expression titer of monoclonal antibody against human CD20 using soy extract, Essential Amino Acid, Non- Essential Amino Acid, Panexin NTS, Peptone, Yeast extract, Insulin-transferrin selenite, Human Serum Albumin, Bovine Serum Albumin, Lipid, and two commercially available supplements, Power and Xtreme feed. In each experiment, the expression level was compared with a well defined media, ProCHO5, RPMI 1640 and DMEM-F12. It has been shown that supplementing the ProCHO5 basal medium with 10% power feed or combination of 5% PanexinNTS,1.5 g/L yeast and 1.5 g/L peptone results in the best production levels with 450 and 425 mg/L of anti CD20 mAb expression level, respectively. Panexin NTS, yeast and peptone cane be proper supplement for fed-batch cell culture instead of commercial Power feed supplement which is a cost effective way to increase expression level. And thereby ProCHO5 may be replaced with common media such as RPMI 1640 and DMEM-F12.

Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues.

Radiation therapy is an effective method used for treatment of many types of cancers. However, this method can cause unwanted side effects such as bone marrow suppression. In this study, the effect of oral administration of biogenic selenium nanoparticles (SeNPs) on total and differentiated white cells profile of BALB/c mice exposed to X-ray radiation was investigated and compared with non-irradiated mice. Sixty female BALB/c mice between six to eight weeks olds were divided into 4 test and control groups in two categories of normal and irradiated mice. In normal mice SeNPs administration was started from the day 0 and followed for a month. Irradiated mice were divided into three groups and were exposed to doses of 2, 4 and 8 Gy. After 72 hr of irradiation, the SeNPs treatment was started and continued for a month. Total and differentiated blood cells counts of both irradiated and non-irradiated groups were monitored during 30 days and the obtained results were compared. Also, the deposition of Se in different tissues and blood serum of normal mice was determined in normal mice after 30 days period of supplementation. In normal mice an increase in the count of neutrophils was observed after 30 days of supplementation. In irradiated mice, SeNPs supplementation led to increase in both lymphocytes and neutrophils counts especially in mice exposed to 2 and 4 Gys radiation. Radiotherapy is categorized as an invasive method which can cause tissue damage and suppress the host immune defense. A restore of lymphocytes which was observed after SeNPs supplementation in irradiated mice can be highly interesting and provide cellular immunity against malignant diseases or other bacterial or fungal infections after radiotherapy.

Production of tissue Plasminogen Activator protein (t-PA) in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming. In this study, reteplase which is the nonglycosylated active domain of t-PA was used to transform TOP10 Escherichia coli (E. coli) bacteria to resolve some of the above mentioned problems. Reteplase cDNA was ligated into pBAD/gIII plasmid which allowed secretion of this protein into the periplasmic space and would allow the correct formation of disulfide bonds in protein structure. The presence of reteplase cDNA in pBAD/gIII plasmid was confirmed by restriction digestion and sequencing. After induction of the expression of this protein by adding 0.0002% L-Arabinose to the medium, the proteins in periplasmic space as well as the inclusion bodies formed inside the cell were extracted. Subsequently, these proteins were purified and detected by Western blot method. Our results showed that the amount of reteplase extracted from periplasmic space was much lower than the extracted inclusion bodies and large quantities of the recombinant protein were present as inclusion bodies. Therefore, it was more efficient to use inclusion body extraction method for protein isolation and purification. We produced active reteplase after its expression in E. coli TOP10 and isolation of inclusion bodies produced the best results for purification and extraction of this protein.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of respiratory tract disease and initiates infection by colonization in nasopharynx. The Haemophilus influenzae (H. influenzae) Hap adhesin is an auto transporter protein that promotes initial interaction with human epithelial cells. Hap protein contains a 110 kDa internal passenger domain called “HapS” and a 45 kDa C-terminal translocator domain called “Hapβ”. Hap adhesive activity has been recently reported to be connected to its Cell Binding Domain (CBD) which resides within the 311 C-terminal residues of the internal passenger domain of the protein. Furthermore, immunization with this CBD protein has been shown to prevent bacterial nasopharynx colonization in animal models. To provide enough amounts of pure HapS protein for vaccine studies, we sought to develop a highly optimized system to overexpress and purify the protein in large quantities. To this end, pET24a-cbd plasmid harboring cbd sequence from NTHi ATCC49766 was constructed and its expression was optimized by testing various expression parameters such as growth media, induction temperature, IPTG inducer concentration, induction stage and duration. SDS-PAGE and Western-blotting were used for protein analysis and confirmation and eventually the expressed protein was easily purified via immobilized metal affinity chromatography (IMAC) using Ni-NTA columns. The highest expression level of target protein was achieved when CBD expressing E. coli BL21 (DE3) cells were grown at 37C in 2xTY medium with 1.0 mM IPTG at mid-log phase (OD600 nm equal to 0.6) for 5 hrs. Amino acid sequence alignment of expressed CBD protein with 3 previously published CBD amino acid sequences were more than %97 identical and antigenicity plot analysis further revealed 9 antigenic domains which appeared to be well conserved among different analyzed CBD sequences. Due to the presence of high similarity among CBD from NTHi ATCC49766 and other NTHi strains, CBD protein expressed here sounds to be theoretically ideal as a universal candidate for being used in vaccine studies against NTHi strains of various geographical areas. Further investigations to corroborate the potency of this protein as a vaccine candidate are under process.

Parathyroid hormone (PTH) secreted by parathyroid glands regulates the metabolism of calcium and phosphorus in bone and kidney. Thereby, it can stimulate bone formation, and is a promising agent in the treatment of osteoporosis. Mature form of PTH consists of 84 amino acids; however, the first 34 residues of PTH cover the majority of hormonal action. In this study, the fusion form of highly soluble rhPTH was expressed at high level in Escherichia coli (E. coli). His6-thioredoxin as an extension for rhPTH improves the solubility of inclusion body. His6-thioredoxin-hPTH (1-34) was ligated into pET32a expression vector. The insertion of 5 amino acids (Asp-Asp-Asp-Asp-Lys) in the N-terminal of PTH made this protein to be digestable specifically by enterokinase enzyme. The fusion form of rhPTH was harvested and purified by immobilized affinity chromatography followed by digestion with enterokinase. Digested rhPTH was purified by applying on size exclusion and ion exchange chromatography to get the highest purity. The mass spectroscopy analysis shows rhPTH molecular weight was 4117.5 Da. The purity was measured by HPLC column which showed more than 97%. Bioassay analysis of rhPTH was performed on rat sarcoma cell UMR-106 in parallel with commercially available rhPTH, Forteo. The result was measured through immunofluorescence detection kit. The data showed that the potency of rhPTH was comparable with commercially available medicine. Thioredoxin was applied as a fusion partner for production of highly soluble rhPTH. This specific fusion partner increased protein solubility and decreased protease reactivity. Purification process was optimized for high recovery and for purity more than 99%. As its biological activity is comparable with marketed drug, this protein is qualified for biopharmaceutical usage.