Early pregnancy factor (EPF) is a pregnancy-associated protein that has immune-suppressive and growth-regulatory activities. It is regarded as a biochemical index by which super-early pregnancy can be determined. The objective of the study was to express the recombinant EPF protein in Escherichia coli (E. coli). The EPF gene amplified by PCR was sub-cloned into a pGEX-6P-1 vector fused with GST gene to construct a recombinant plasmid. The recombinant plasmid was then transformed into E. coli BL21.The expression of recombinant protein was analyzed by SDS-PAGE and Western blot after induction by IPTG. The recombinant protein was purified by high-affinity GST resin chromatography. The prokaryotic expression plasmid pGEX6P-EPF for producing EPF protein was constructed and the recombinant protein with a molecular weight of 37.3 kDa was expressed successfully in E. coli, which was recognized by the anti-GST monoclonal antibody in Western blot analysis. The recombinant EPF protein was obtained after GST affinity purification.

Following the LAMP protocol,a set of four specific primers was designed based on the sequence of gene encoding envelope protein 28（VP28） of WSSV.Using the recombinant plasmid of pMDT-VP28 as a standard template,the LAMP method for WSSV detection was developed by optimizing its reaction system and conditions.Its detection limit for WSSV was determined by detecting the ten-fold diluted WSSV-DNA（107 to 10-3 copies/μL） and compared with nested PCR.The optimized reaction temperature and time of WSSV-LAMP were at 65 ℃ for 60 min,respectively.Detection limit of the LAMP method for the WSSV-DNA was determined to be 10 copies/μL,whereas that of nested PCR was 100 copies/μL,suggesting the LAMP method was much more sensitive than the nested PCR methods for WSSV detection.In conclusion,the LAMP method for WSSV detection is more convenient,rapid,and sensitive than the nested PCR,and do not need the expensive sophisticated instrument.Therefore,it is very suitable for detection in the fields that would provide a novel rapid detection approach for early WSSV infection in aquaculture.

目的:构建缺失免疫抑制位点的人早孕因子基因的原核表达载体pGEX6P-EPF11,诱导GST-EPF11融合蛋白在大肠杆菌中表达,并免疫小鼠,测定效价.方法:以pMD18T-HSPE1模板,PCR扩增缺失免疫抑制位点的人早孕因子基因片段,经限制性内切酶BamH I和Xho I双酶切后,连接到原核表达载体pGEX-6P-1中,构建重组表达质粒pGEX6P-EPF11.将表达质粒转化大肠杆菌BL21,以1 mmol/L IPTG 进行诱导表达GST-EPF11融合蛋白,Western blot鉴定.以表达的GST-EPF11融合蛋白作为免疫原免疫小鼠,抗体效价用ELISA检测.结果:在大肠杆菌中成功表达出相对分子质量约35 000的融合蛋白GST-EPF11.ELISA法检测免疫小鼠的抗体血清可达1:12 800.结论:在大肠杆菌中成功表达出GST-EPF11融合蛋白,并免疫了小鼠,测定了抗体效价,为下步制备缺失免疫抑制位点的人早孕因子的单克隆抗体(mAb)奠定了基础.

RT-PCR was used to study the expression of heat-shock 10 kDa protein.The results showed that there were two different HSP transcriptons of varied sizes in Homo liver cancer SMMC-7721 and Mus NS0.One HSP cDNA was composed of 312 bp(named HSPE1-1) and held 99% homology with HSPE1 mRNA reported(NM-002157).The other HSP gene cDNA was consisted of 313 bp(named Hspe1-1) and had 97% homology with Hspe1 mRNA reported(NM-008303).The homology between HSPE1-1 and Hspe1-1 was 92% in nucleotide sequence and 97% in amino acid sequence.Prokaryotic expression plasmids pET-28a-HSPE1-1 and pET-28a-Hspe1-1 for producing HSPE1 and Hspe1 proteins were constructed successfully.Its molecular weight was 14 kDa.Anti-His6 monoclonal antibody was used to demonstrate the identity of the proteins in Western Blot.The corresponding proteins were obtained after purification by Ni-NTA chromatography.The purified corresponding proteins would be helpfulin further studies of their molecular structure,biological function and clinical application.

The objective of the present study is to establish the Marc-145 cell line that stably expresses porcine poFcγRⅢ(porcine Fc gamma receptor Ⅲ,poFcγRⅢ).The total RNA was extracted from porcine alveolar macrophage(PAM),and poFcγRⅢ and γ-chain cDNAs were cloned by RT-PCR,then they were inserted into the eukaryotic expression vectors pcDNA3.1(+)and PIREShyg3 respectively.The marc-145 cell line was stably transfected with pcDNA3.1-poFcγRⅢ and PIREShyg3-γ plasmids by Lipofectamine2000,then the co-transfected cells were selected by HygromycinB(300 mg/L)and G418(400 mg/L).The expression of poFcγRⅢ on transfected cells was verified through RT-PCR,rosetting test and FCM.The eukaryotic expression vectors were successfully constructed and the Marc-145 cell line with stable poFcγRⅢ expression was obtained.The Marc-145 cell transfected with the poFcγRⅢ cDNA were able to bind porcine IgG.

【Objective】 The objective of this experiment is to develop an immunochromatographic strip for rapid detection of neomycin(NEO).【Method】 The monoclonal antibodies(mAb) against neomycin were generated and labeled with colloidal gold particles.Then the immunochromatographic strips for detection of NEO were developed and identified.【Result】 Ka of NEO monoclonal antibody was 3.75×1010 mol·L-1.The detection limit of the strip was 50 ng·mL-1 by naked eyes.The cross-reactivity with other antibiotics,such as gentamicin,streptomycin,tobramycin,sisomicin,ractopamine,salbutamal,tylosin,chloramphenic,was not detected.In parallel experiments,the test strip gave the same results as HPLC-MS-MS did.【Conclusion】 An immunochromatographic strip test method was established and the strip can be used to detect NEO residues rapidly with high specificity,sensitivity and simplicity in different samples.