以大肠杆菌为载体进行蛋白表达的工艺研发虽然技术成熟,但由于表达产物不同,所需要的工艺研发存在很大差异.下游纯化工艺的好坏对于疫苗产量、质量、效果有着直接影响.本文探讨了以包涵体形式表达的基因工程疫苗下游工艺,以供参考.

[Objective] To establish an indirect ELISA method for the antibody titer of peste des petits ruminants (PPRV) N protein suitable for PPR epidemic prevention.[Method] N5 gene was amplified based on the published N gene sequence of Nigeria 75/1 strain in GenBank,and the amplified DNA fragment was inserted into the expression vector pET-32a(+) to construct the recombinant plasmid pET-32a-N5.The resulting protein was expressed in TransB (DE3) chemically competent cell by IPTG induction and purification.SDS-PAGE analysis showed that the protein was soluble,and the immunological activity of the target protein was proved by Western-blot identification.[Result] An indirect ELISA method detecting antibody of PPRV N using purified PPRV N protein as a coated antigen.Antibody of 112 goats serum samples were detected collected from infected non-plague area,and the accordance rate reached 94.8% compared with C-ELISA.[Conclusion] The method showed good sensibility,repeatability and specificity.

[目的 ]选择一种适合口蹄疫基因工程疫苗生产的佐剂.[方法 ]通过ISA 206和ISA201 VG油佐剂乳化后物理性状检验,参照现有口蹄疫灭活疫苗乳化工艺,选用ISA 206和ISA201 VG油佐剂,分别用4.6:5.4、1:1、5.4:4.6的水相和油相比例进行乳化,制备疫苗,于2~8℃保存15个月,分别于第3、6、9、12、15个月取样,进行疫苗物理性状检验.[结果 ]ISA201VG佐剂疫苗物理性状好于ISA 206油佐剂;疫苗水相和油相最佳配制比例为1:1;疫苗的外观、剂型、稳定性和黏度均符合产品的质量要求.[结论 ]ISA201VG佐剂可以作为口蹄疫基因工程疫苗佐剂使用.

Based on the published gene sequence of the N gene sequence of Nigeria 75/ 1strainwas synthesized in GenBank, two pairs of specific primers were designed for amplifying the gene of N1and N5from N gene, connection to the N1and N5gene with Overlap-extension PCR, The sequencing of the N1-N5gene and the expression vector pET-32a(+) by EcoRI and NotI restriction enzyme digestions and inserted into the expression vector pET-32a(+) to construct the recombinant plasmid pET-32a-N1-N5. The recombinant protein was expressed in TransB(DE3) Chemically Competent Cell by IPTG induction and purified.SDS-PAGE showed that the protein was proved to be soluble and Western-blot analysis indicated that the target protein was proved to be immunological activity, The expressed N1-N5protein could be used for the preparation of a rapid surveillance of antibody level of PPRV.

A tapeworm, Taenia solium, remains a great threat to human health, particularly in developing countries. The life cycle of T. solium is thought to be terminated via vaccination of intermediate hosts. In this study, we constructed a recombinant attenuated Salmonella typhimurium live vaccine strain χ4558 expressing a TSOL18 antigen. SDS-PAGE and Western blot confirmed the expression of the interest protein and its antigenic property. The recombinant strain stably propagated in vitro, of which the growth was not reversely influenced by TSOL18 protein expressed. It was also shown that mice survived 1012 CFU of S. typhimurium χ4558, while all mice infected with 107 CFU of the wild-type died within five days. The mouse experiment indicated that vaccine strain χ4558 induced a high titer of specific antibody for a long time. In contrast to the controls, the vaccinated mice had an obvious augment of CD4+ and CD8+ T lymphocytes and the percentage of helper CD4+/CD8+ T lymphocytes was significantly increased (p < 0.01). After oral administration, S. typhimurium χ4558 was first colonized mainly in the Peyer's patches and then predominantly in the mesenteric lymph nodes and spleens in the vaccinated mice. In addition, the high levels of specific anti-TSOL18 antibodies were also observed in pigs administrated with S. typhimurium χ4558. Collectively, these results demonstrate the possibility of use of an attenuated S. typhimurium strain as a vector to deliver protective antigens of T. solium.

In this paper,using four varieties of sweet potato 'Xusu18,Xusu22,Xusu25,Xusu27' as tested materials,the effect of SPVD on sweet potato yield were quantitatively analyzed by comparing the tuberous root yield of sweet potato plants with and without symptoms of the disease.The results showed that the disease index of four varieties of sweet potato disease-free seedlings was 18.06 %,0.22 %,5.81 % and 54.56 %,respectively,while seedlings with SPVD symptoms was 82.27 %,88.14 %,78.09 % and 81.42 %,respectively,35 d after planting.In the harvest period,the yield losses of four sweet potato varieties were 77.84 %,80.14 %,68.16 % and 31.06 %,respectively.

In this study,the resistances of 81 sweet potato germplasm resources to sweet potato rot nematode,black rot and root rot were identified and evaluated.The results indicated that very few sweet potato varieties(lines) could simultaneously resist these three diseases,only Zheng 04-4-2 and Xu 062826 had high resistances not only to the invasion and expansion of rot nematode,but also to black rot and root rot.In addition,some tested resources such as Taizhong No.7,Zhezi No.1 and Xu 060407 had the double resistances,and they can be used as the double-resistant parents in the disease-resistant breeding of sweet potato.

In this study,triumph 100 seedlings were inoculated with Ceratocystis fimbriata at the fifth day after silicon treatment,and disease indexes were determined per 24 hours.The results showed that silicon treatment could prolong the incubation period of C.fimbriata and significantly decrease the incidence and disease index of sweet potato black rot.Therefore,silicon treatment could reduce the damage of black rot to sweet potato,and improve the disease resistance of sweet potato varieties.

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.

Porcine reproductive and respiratory syndrome virus(PRRSV) nucleoprotein gene,amplified by reverse transcription-PCR was cloned into pGEM-T easy vector,After sequencing,the N gene was blasted with PRRSV genome sequence downloaded from GenBank with DNAstar software.The results indicated that the homologus nucleotide sequence of N of PRRSV-GS was 93.3 % and 66.1 % compared with PRRSV-VR2332 and LV.The N gens was subcloned into prokaryotic expressing vector pGEX-4T-1,the recombinant plasmid named pGEX-4T-1-N was constructed.The pGEX-4T-1-N was used to transform into E.coli BL21(DE3).The results of SDS-PAGE showed that the N gene was expressed in a high level as a 40 ku.

【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.

Objective The biosafety and tissue distribution of recombinant expression plasmid pVAX1/TSOL18,an anti-cysticercosis DNA vaccine,was investigated in mice. Methods Recombinant pVAX1/TSOL18 plasmid was intramuscularly injected on the legs of mice,which were killed after 1 d,5 d,15 d and 30 d post-injection and the gDNA were extracted from hearts、liver、spleen,etc.at the different time.The distribution and the residual time of recombinant pVAX1/TSOL18 plasmids in the tissue of mice were analyzed by PCR technology.At the same time,the feces of mice were tested by PCR to investigate the release of pVAX1/TSOL18 in the external environment. Results One day and five days after immunization,the target fragments were amplified from the different gDNA of mice tissues.To the different individual,the tissue are also different which the target fragment were amplified from,but from blood of all the injected mice,the target fragment were obtained.Contrast to the result on the first day,the fragment are darker than that on the fifth days.Fifteen days after immunization,there was only one mouse in which the fragment of plasmid TSOL18 gene could be detected.Thirty days later,no any recombinant plasmid could be detected in all the mice.And the TSOL18 plasmid gene were not detected at the first and fifth day in the feces. Conclusion Recombinant plasmid pVAX1/TSOL18 is safe to mice and the surrounding environment as a vaccine.

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.