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The Expression Quantity With Vector-receptor Correlation Studies Of RhIL-1in P.Pastoris

Posted on:2015-12-01Degree:MasterType:Thesis
Country:ChinaCandidate:Y J YangFull Text:PDF
GTID:2180330431978515Subject:Cell biology
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There are11members in Interleukin-1(IL-1) family mainly including interleukin-1α (IL-1α)、interleukin-1β (IL-1β) and interleukin-1receptor antagonist (IL-1Ra),which are the key ingredients ofcytokine network.. IL-1has a very wide range of biological activities,for example, activating thehemopoietic system and immune system,stimulating lymphocyte proliferation, inducing the antitumoractivity of mononuclear cells、macrophages and natural killer cells. Therefore this kind of proteinexpression with the genetic engineering technology, has an important significance and application value.At present, the commonly used Pichia pastoris strains that express recombinant proteins are: GS115、KM71、X–33、SMD, etc. The common types of excretive expression vector are: pPIC9K、pPIC9、pPICZαA, etc. To study the effect of different carrier–receptor combinations to rhIL-1expression, thisarticle select three genes:IL-1alpha, IL-beta and IL-1ra,three receptors:GS115,SMD1168and X-33,three carriers:pPIC9K,pPIC9and pPICZαA, which based on the principles of permutation andcombination to build their engineering bacteria and test their best expression.The results show that, for the expression of rhIL-1α, the combination of X-33/pPICZαA had thehighest quantity, about0.346mg/L. For the other: X-33/pPICZαA﹥GS115/pPICZαA﹥GS115/pPIC9K﹥GS115/pPIC9﹥SMD1168/pPICZαA﹥SMD1168/pPIC9K﹥SMD1168/pPIC9.For rhIL-1β,X-33/pPICZαA combination had the highest expression quantity, about0.553mg/L. For the other:X-33/pPICZαA﹥GS115/pPICZαA﹥GS115/pPIC9K﹥GS115/pPIC9﹥SMD1168/pPICZαA﹥SMD1168/pPIC9K﹥SMD1168/pPIC9.The rhIL-1Ra has the same phenomenon, X-33/pPICZαAcombination had the highest expression quantity, about0.385mg/L. For the other: X-33/pPICZαA﹥GS115/pPICZαA﹥GS115/pPIC9K﹥GS115/pPIC9﹥SMD1168/pPICZαA﹥SMD1168/pPIC9K﹥SMD1168/pPIC9Compare the best expression quantity of rhIL-1α, rhIL-1βand rhIL-1Ra we found:that theexpression of rhIL-1β>The expression of rhIL-1Ra>The expression of rhIL-1αin X-33/pPICZaAThe results show that: The expression quantity of rhIL-1is closely related to the carrier-receptor combination, The combination of X-33/pPICZaA was the strongest to express the three members of IL-1,SMD1168/pPIC9combination was the weakest. On the other hand, the expression quantity of rhIL-1isalso associated with the types of IL-1, rhIL-1βexpression quantity was the highest, rhIL-1Ra second,rhIL-1αslightly less than the amount of rhIL-1Ra.
Keywords/Search Tags:Pichia pastoris, engineering bacteria, Expression vector, recipient bacterium, Humanrecombinant interleukin1
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