The expressive vector of pGEX-Tsol18-sp was constructed using the genetic modified Tsol18 gene of Taenia solium onchospere by the site-directed mutation of 4 bases in its coding sequence and removing of the sequence fragment encoding its N-terminal signal peptide.After induced with IPTG,the expressed proteins of TSOl18 and TSOl18-sp were probed by SDS-PAGE,Western blot and its stability was evaluated by storing at 4 ℃.The effects of immuno-protection of these two recombinant proteins were compared by ELISA and counting of Cysticercus in the muscle tissue of experimental pigs.The results revealed that the modified Tsol18-sp,in which 7 bases were synonymously mutated,could be expressed in the recombinant pGEX-Tsol18-sp vector.The soluble recombinant protein with a molecular weight of 38.7 ku can be probed by the specific serum against Taenia solium oncosphere,and only 20.1% was degradated in storage at 4 ℃ for 2 months.Both of the recombinant proteins can induce high titre of antibodies in the vaccinated pigs,but the recombinant protein TSOL18-sp showed a better protection rate(100%) against the challenge of Cysticercus cellulose than TSOL18 revealed(20%) by the reduced counts of Cysticercus in the muscle tissue of vaccinated pigs.It was suggested that the TSOL18-SP is a effective and stabile antigen,and could be used as a potential antigen for the immunoprophylaxis of Cysticercosis cellulosae.

【Objective】To construct a recombinant attenuated Salmonella typhimurium live vector strain expressing the antigen of Taenia solium oncosphere 18.【Method】The Taenia solium oncosphere 18 gene was cloned and reconstructed into expression vector pYA3341.The recombinant plasmid pYA3341-TSOL18 was finally electro-transformed into an attenuated S.typhimurium definitive host strain of X4500 and the recombinant strain of X4500(pYA3341-TSOL18) was identified its immunogenicity of expression protein,stability,growth curve,safety in vitro and immunity evaluation by animal experiment in mouse.【Result】With restriction enzyme digestion and gene sequencing,it was showed that the recombinant plasmid has been constructed successfully.Urea-SDS-PAGE and Western blot were used to detect the new protein band and its antigenic property in S.typhimurium X4500(pYA3341-TSOL18).The recombinant strain could stably propagate at noruishment selection pressure in vitro and its growth wasn’t influenced by the expression protein on the whole and its safety and reliability were verified by mouse experiment.The destinated antibody was detected by ELLISA.【Conclusion】Successfully constructed A recombinant attenuated S.typhimurium strain X4500(pYA3341-TSOL18) was succefully constructed,an oral administration live vaccine which can express TSOL18 protein stably.

There are two kinds of embryonic stem cells.One is embryonic stem cell(ESC) from inner cell mass(ICM)of forepart embryo,and the other is embryonic germ cell(EGC)from primordial germ cells(PGCs)of embryonic gonad.PGCs are precursor cells of primordial germ cell and antecedent cells of sperm and ovum.In 1992,PGCs were isolated firstly from mouse post-implantation embryo,which was also approved as material of embryonic stem cells.Many animals' PGCs,from human,mouse,pig and chicken,have been isolated and cultured and attained EGC.In this paper,kesearches on avian EGC cell morphology and migration,isolation,culture and identification,were analyzed and summarized.

Mice were immunized with the constructed recombinant attenuated Salmonella typhimu-rium live vector vaccine X4550(pYA3341-TSOL18) to evaluate the efficacy of the vaccine.The mouse sera IgG and IgM antibodies and the mouse liquor entericus secreting IgA antibody were examined by ELISA.The recombinant vaccine bacteria persisting in mice immune tissues were examined.The splenocyte subsets were assayed.The IgM antibody could be detected on day 7 post-immunization(PI) and the D490 nm of IgG antibody could reach to 0.645 on day 28 post-2nd-immunization(P2I) and the secreted IgA could be detected in mouse liquor entericus on day 42 P2I.The vaccine could induce production of the antibodies including IgM,IgG and IgA.The vaccine could be detected in mice spleen on day 21 PI.Both the amounts of CD4+ and CD8+ T lymphocytes and the proportion of CD4+ to CD8+ T lymphocytes were increased significantly in the mice immunized with the vaccine X4550 as compared with those in the mice of control groups,indicating that the expression of recombinant TSOL18 protein did not affect ecesis of the recombinant vaccine.The results revealed that the vaccine may build a immunifaction by elevating the proportion of CD4+/CD8+ T cells and could induce Th1 and Th2 immunity responses at the same time.

In order to improve the biologic activity and stability of the recombinant 18 kD antigen(TSOL18) of Taenia solium onchospere,the expressive vector of pGEX-TSOL18-SP was constructed with the modified T.solium onchospere TSOL18 gene by the rite-directed mutagenesis of 4 bases in its coding sequence and removing of the sequence fragment encoding its N-terminal signal peptide.After induced with IPTG,the expressed proteins were analyzed by SDS-PAGE and Western-blot.The rabbits were immunized with the purified TSOL18-SP protein and the titer of serum antibody was detected by agar double diffusion test.It was shown that seven bases were mutated in the reconstructed TSOL18 gene with 345 bp open reading frame.Compared with the published sequences of 18 kD protein of T.solium oncosphere(GenBank accession number AF017788),its relative similarities in nucleotide and amino acids were 97 % and 100 %,respectively.Molecular mass of TSOL18-SP was approximately 38.7 kD on SDS-PAGE and reacted well with mice positive serum against T.solium onchospere.The antibody titers of rabbit against TSOL18-SP reached up to 1∶32～64 by agar double diffusion test.The results indicated that the TSOL18-SP protein has a better immunogenicity and it could be used for the vaccine development of cysticercosis.

Embryonic stem cell(ESC)could be self-renewal incessantly in long term in vitro.It had high differentiated potentiality and could differentiate into almost every type of cell of foetus and adult.Such as heart muscle cell,nerve cell,epithelia,liver cell,blood corpuscle,islet cell,lipocyte and germ cell.So ESC could he applied widely in cell therapy,tissue and organ replacement therapy,devel- opmental biology and other researches.At present a variety of cell types had be reported that were induced by directed differentiation in vitro.This paper analyzed and summarized infection factors of induced differentiation and several major cell types induced from ESC, and to offer reference for ESC's induced differentiation research.

The full-length P32 gene and the truncated P32 gene (MP-32) were amplified from the recombinant plasmid pMD-P32 by polymerase chain reaction (PCR) and cloned into pcDNA3. 1(+) and pcDNA3.1-CpG respectively. The recombinant plasmids (pcDNA3.1-P32, pcDNA3.1-CpG-P32 and pcDNA3. 1-CpG-MP32) were transfected into BHK-21 cells by using lipofectin. The expressed P32 protein was confirmed by indirect immunofluorescence assay (IFA). The BALB/c mice were immunized with these recombinant plasmids by intramuscular injection. The specific antibodies aginst CPV were detected by ELISA kit weekly. The murine splenic T lymphocyte subgroups CD4+ and CD8+ were detected by flow cytometry. Results showed that the P32 protein was expressed successfully in vitro. After 2 weeks post im munization, the specific IgG antibodies against CPV were detected in the vaccinated mice. The percentage of CD4+ /CD8+ T cells was significantly higher than that of the control. In conclusion, these constructed eukaryotic vectors could induce humoral and celluar immune responses in mice.

An unknown gene,SLC10,was cloned by RT-PCR from Cysticercus cellulosae on the base of trans-spliced leader sequence.The SLC10 gene was cloned from vector of pGEM-SLC10 and inserted into pPIC9k to construct a recombinant secretory vector named pPIC9K-SLC10.Then the pPIC9k-SLC10 was transformed into E.coli JM109.The recombinant vector was identified by PCR,enzymatic analysis and sequencing.The positive plasmid was linearized with Sac I and transformed into GS115 by electroporation and the insert was integrated into genomic DNA of Pichia pastoris by means of homologous recombination.The recombinants with multiple integrated copies of SLC10 were screened using gradient concentration G418 and induced with methanol.The results of SDS-PAGE and Western-blot showed that the expressed protein was not of immunoreaction.

【Objective】Cloning, expression and immunohistochemistry of SLC10 gene in Taenia solium are not only helpful to us to understand the biological characteres of Taenia solium but also helpful to us to find the vaccine or diagnostic candidates. 【Method】The unknown gene was cloned by spliced leader-based PCR from Taenia solium. The recombinant SLC10 protein was expressed in E. coli in a fusion pattern and purified by affinity chromatography. Using purified recombinant SLC10 protein, ELISA and immunohistochemistry tests were made.【Result】The new gene was named SLC10. The ORF of SLC10 was found to be 507 bp, encoding an 18.2 kD protein. The genomic DNA of SLC10 was found to be 1 107 bp consisted of two exons and one intron. ELISA results showed that all detected sera, including 70 normal and 75 cysticercosis sera, did not react with purified recombinant SLC10 protein. Immunohistochemistry showed that the native SLC10 protein distributed mainly in inner cyst walls but not in the scolex in C. cellulosae. 【Conclusion】 Together with predicted results, it is suggested that the SLC10 protein is a non-secretory structural protein, not involved in induction of the host’s immune reactions against infection at least at the larval stage.

Comprehensive research of stem cells which is one of the energiest,most impacting and applied foreground research directions in life science,when united with genetic engineering and transplantation may provide significance of theory and practice.Feeder cells have a great impact on isolation and culture of ES cells.Refering to plenty of information,the author combined with experiment experience explained the general impact of feeder cells on embryonic stem cells,introduced the class of feeder cells and discussed the influencing factores of isolation and culture.

Comprehensive research of poultry stem cells united with genetic engineering and transplantation may provide significance of theory and practice. This paper introduced the general study background of poultry stem cells and embryonic germ cells,discussed the influencing factoes of isolation and culture,and explained the identification of cell biological characteristics. In the meanwhile,its applications and future use of science and technology are prospected.

近几年,生物技术在全世界范围取得了飞速的进展,一些重大生物研究项目如人类基因组计划、克隆技术等开始引起公众的广泛注意.本文综述了二十世纪九十年代以来基因组生物技术的发展状况,重点介绍了人类基因组计划、动物基因组程序,以及基因组学研究成果的应用.

生物技术在全世界范围取得了飞速的进展,与此同时其应用和产业化在各国政府、科研机构和生物技术公司的大力参与和激烈竞争中也逐步加快,预计它将成为许多国家经济的重要支柱产业之一.一些重大生物研究项目如人类基因组计划、克隆技术等开始引起公众的广泛注意.综述了20世纪90年代以来动物生物技术的发展状况,对基因组研究、转基因动物、克隆技术和细胞培养等重要研究方向作了介绍和分析.