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Construction And Expression Of The Fusion Gene Of Somatoatatin (S14) And Hepatitis Surface Antigen (HBsAg) In Pichia Pastoris

Posted on:2003-02-14Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z Z WangFull Text:PDF
GTID:1100360092966700Subject:Genetics
Abstract/Summary:PDF Full Text Request
Somatostatin is a neurohormone which p1ay mu1tilatera1physio1ogic function in vertebra animal body (including human ), tostudy ss genetic engineering vaccine is of great significance andhave extensive appl ication potent ial.The present study involves in the folling contents:1. Synthesis of Sl4 gene:According to the sequence of S,. gene, S,. gene fragment wassynthesized and cloned after a pair of primers was designed. DNAfragment was inserted into pBluescript Vector, and transformed intoE. coli. N109, Recombinants were grew in LBA+culture medium plateand screened. P1asmid DNA was mp1ified by using RCR andidentified by sequening. The results sho'ed that the sequence ofSl4 gene is identical with prior designation.2. AInPlification and c1one of HBsAg gene:A pair of primers wes designed, IIBsAg gene,as mplified byusing PCR from PIIBV. HBsAg gene fragnent,as inserted intopBluescript vector and transformed into N109. Plasmid was5@)llX*#1#&texscreened by quickly phenol method and identified by shanghaijikang Bio--engineering Co. Ltd. The results showed it has a rightDNA fragments.3. Fusion of S14 and HBsAg gene:Sl4 and HBsAg gene were obtained from that pB1uescript S.. andpB1uescript HBsAg were digested by S8cII and SacI enzyme separatelyand were linked under T4 DNA ligase, then pBluescript S,,/HBsAgplasmid was constructed and transformed into JM109, andrecombinants were screened. DNA band is about 600 bp when p1asmidwas digested by SacII and SacI enzyme, and is equal to that of HBsAggene; but it is about 700bp when plasmid was digested by XhoI andSacI enzyme, and is equa1 to the weight of S,./HBsAg fusion gene. Theresults showed that S14 and HBsAg gene was fused proper1y inpBluescript vector -4. Sub--clone of S,./HBsAg fusion gene:pBuescriptS,./HBsAg and pPICZaA were digested separately byXhoI and XbaI enzyme, and were linked under T4 DNA Ligase, pPICZaAS,,/HBsAg was constructed and transformed to E. coli. T0P10, therecombinant was digested by XhoI and XbaI, SacII and XbaIseparately and e1ectrophoresis test, the resu1ts showed S,./HBsAgfusion fragthent 'as inserted into pPICZaA Vector.5. Construction and expression of yeast engineering yaccine:S14/gnsAg 'as transfOrmed to yeast host straln X33 by meansof electroporation after pPICZaA S,.aresAg 'as l ined by SacI enzyme.The single fungus,as choose and dibble inocu1ating in We and amplate, The positive fungus was gro'ing in rm but not in W, and was6inoculated in YPD which included Zeocine 500ug/ml and 1000ug/ml.5 transformers were amPl if ied by PCR, three is same with positivecontrol. It is showed that yeast host strain X--33 included S../HBsAgfragment. one was named for P. pastoris X--33/S,./HBsAg. P. pastorisX--33/S../HBsAg was expressed under the induction of 0. 5$methanol.SDS--PAGE showed that after being induced with 0. 5% methano1 for 3d,the expressed products existed in supernatant and it's molecularweigh was about 22KD, Western b1ot showed good antigenicity andspeci ficity of expressed product.6. Anima1 experiment:Rabbits and 10d old mice were inoculated separately with thesupernatant from the cu1ture of yeast cell transformed withpPICZaASl4/HbsAg, which were induced with 0. 5$methano1. S,.Antibodies were monitored at interal of one week, the results showedthat it is Positive between 2 to 4 weeks after inoculation; themice were weighed every 5 day, the results showed that weightdifference is significant between the inoculated and controlgroup. Seroreaction of test group is positive using HBsAg ELISA kit,but control group is negative.These study results showed that S,,/HBsAg fusion protein wassuccessful1y expressed in P. pastoris first. The study laysfoundation for further industria1 production and application ofc1inical medicine and aniasl husbandry.
Keywords/Search Tags:Somatostatin, HBsAg, Pichiapastoris, gene expression
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